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Identification of Caspase-1 and Caspase-3 Substrates
And Study on Caspase-1 Substrates in Glycolytic Pathway
Wei Shao
Department of Biochemistry
McGill University
Montreal, Quebec, Canada
August 2007
A thesis submitted to the faculty of Graduate Studies and Research in partial
fulfillment of the requirements for the degree of Master of Science
© Wei Shao, 2007
Identification of Caspase-l and Caspase-3 Substrates ,
And Stndy on Caspase-l Snbstrates in Glyçolytic Pathway
Wei Shao
Department of Biochemistry
McGill University
Montréal, Québec, Canada
August 2007
A thesis submitted to the faculty of Graduate Studies and Research in partial
fulfillment of the requirements for the degree of Master of Science
© Wei Shao, 2007
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Bibliothèque et Archives Canada
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Direction du Patrimoine de l'édition
395 Wellington Street Ottawa ON K1A ON4 Canada
395, rue Wellington Ottawa ON K1A ON4 Canada
NOTICE: The author has granted a nonexclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distribute and sell theses worldwide, for commercial or noncommercial purposes, in microform, paper, electronic and/or any other formats.
The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
ln compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis.
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ABSTRACT
Apoptosis is executed by caspase-mediated cleavage of various proteins. Elucidating the
consequence of substrate cleavage provides us with insight into cell death and other
biological processes. In this study, we applied the diagonal gel approach, a proteomic
strategy, to identify substrates of the inflammatory caspase, caspase-1 and the cell death
executioner caspase, caspase-3. Our results showed significant overlap between the
substrates cleaved by both caspase-1 and - 3 . Such substrates are implicated in common
cellular functions, including maintenance of the cytoskeleton, folding of proteins,
translation, glycolysis, bioenergetics, signaling and trafficking. An important finding is
that many glycolysis enzymes were targeted specifically by caspase-1. Processing of
these glycolysis enzymes by caspase-1 was confirmed by cleaving in vitro transcribed
and translated substrates with recombinant caspase-1. We have focused our further
analysis on certain glycolysis enzymes. We have characterized the caspase-1 cleavage
site in GAPDH. Point mutation of the Aspartic acid at position 189 to Alanine (D189A)
in GAPDH blocked its cleavage by caspase-1. In vivo, in a mice model of septic shock,
characterized by hyperactivation of caspase-1, we observed depletion of the full-length
forms of these glycolysis enzymes in the diaphragm muscle. Further studies in caspase-1
deficient mice will confirm whether this depletion, in caspase-1 proficient mice, was due
to caspase-1 processing of the glycolysis enzymes. This provides a direct link between
caspase-1 activation and inhibition of glycolysis, which might have important
implications on loss of muscle contractility in septic shock.
ii
ABSTRACT
Apoptosis is executed by caspase-mediated cleavage of various proteins. Elucidating the
consequence of substrate cleavage provides us with insight into cell death and other
biological processes. In this study, we applied the diagonal gel approach, a proteomic
strategy, to identify substrates of the inflammatory caspase, caspase-1 and the cell death
executioner caspase, caspase-3. Our results showed significant overlap between the
substrates cleaved by both caspase-1 and -3. Such substrates are implicated in common
cellular functions, including maintenance of the cytoskeleton, folding of proteins,
translation, glycolysis, bioenergetics, signaling and trafficking. An important finding is
that many glycolysis enzymes were targeted specifically by caspase-l. Processing of
these glycolysis enzymes by caspase-1 was confirmed by cleaving in vitro transcribed
and translated substrates with recombinant caspase-1. We have focused our further
analysis on certain glycolysis enzymes. We have characterized the caspase-1 cleavage
site in GAPDH. Point mutation of the Aspartic acid at position 189 to Alanine (D189A)
in GAPDH blocked its cleavage by caspase-l. In vivo, in a mice model of septic shock,
characterized by hyperactivation of caspase-1, we observed depletion of the full-length
forms of these glycolysis enzymes in the diaphragm muscle. Further studies in caspase-l
deficient mice will confirm whether this depletion, in caspase-1 proficient mice, was due
to caspase-1 processing of the glycolysis enzymes. This provides a direct link between
caspase-l activation and inhibition of glycolysis, which might have important
implications on loss of muscle contractility in septic shock.
11
RESUME
L'apoptose ou la mort cellulaire programme est un phenomene medie en partie par les
caspases capables de cliver plusieurs substrats. Elucider le role et les consequences du
clivage de ses differents substrats nous fournira plus elements dans la comprehension de
la mort cellulaire et autres processus biologiques. Dans cette etude, nous avons applique
l'approche du gel diagonale, une strategie proteomic, afin d'identifier tous les substrats
clives par la caspase inflammatoire, caspase-1, et les compares aux substrats clives par la
caspase apoptotique, caspase-3. Nos resultats montrent dans un premier temps, qu'un
nombre considerable de substrats est clive a la fois par caspase-1 et aussi par caspase-3,
cela appuie leur commun dans des fonctions cellulaires, y compris l'entretien du
cytosquelette, conformation proteique, la traduction, la glycolyse, bioenergie, la
signalisation et le trafique cellulaire. Dans notre approche, nous avons remarque que
plusieurs enzymes impliquees dans la glycolyse sont des substrats uniques de caspase-1.
Le clivage de ces enzymes de glycolyse par caspase-1 a ete confirme in vitro en presence
de la proteine recombinante caspase-1. Nous avons concentre notre analyse
supplemental sur certaines enzymes telle que GAPDH. Nous avons pu caracterise le
site de clivage de la GAPDH par capase-1, et en realisant une mutation cible de l'acide
Aspartique de la position 189 en Alanine (D189A), on aboutit a un blocage du clivage par
caspase-1. Nos resultats in vitro ont ete completes par une approche in vivo en utilisant un
model animal de choc septique, une souris caracterise par une hyperactivation de
caspase-1, nous avons pu observe une decroissance dans les formes longues de ces
enzymes dans le muscle du diaphragme. L'utilisation de souris defectueuses en caspase-1
vont nous demontrer si le clivage et la diminution dans la forme longue de ces enzymes
est reellement le resultat d'un clivage par caspase-1. Cela fournira un lien direct
entre 1'activation de caspase-1 et 1'inhibition de la glycolyse qui pourra avoir des
percussions importantes sur la perte de contractilite du muscle dans le choc septique.
iii
RÉSUMÉ
L'apoptose ou la mort cellulaire programme est un phénomène medié en partie par les
caspases capables de cliver plusieurs substrats. Élucider le rôle et les conséquences du
clivage de ses différents substrats nous fournira plus éléments dans la compréhension de
la mort cellulaire et autres processus biologiques. Dans cette étude, nous avons appliqué
l'approche du gel diagonale, une stratégie proteomic, afin d'identifier tous les substrats
clives par la caspase inflammatoire, caspase-1, et les comparés aux substrats clives par la
caspase apoptotique, caspase-3. Nos résultats montrent dans un premier temps, qu'un
nombre considérable de substrats est clivé a la fois par caspase-1 et aussi par caspase-3,
cela appuie leur commun dans des fonctions cellulaires, y compris l'entretien du
cytosquelette, conformation protéique, la traduction, la glycolyse, bioénergie, la
signalisation et le trafique cellulaire. Dans notre approche, nous avons remarque que
plusieurs enzymes impliquées dans la glycolyse sont des substrats uniques de caspase-1.
Le clivage de ces enzymes de glycolyse par caspase-1 a été confirmé in vitro en présence
de la protéine recombinante caspase-1. Nous avons concentré notre analyse
supplémentaire sur certaines enzymes telle que GAPDH. Nous avons pu caractérisé le
site de clivage de la GAPDH par capase-1, et en réalisant une mutation cible de l'acide
Aspartique de la position 189 en Alanine (D 189A), on aboutit a un blocage du clivage par
caspase-1. Nos résultats in vitro ont été complétés par une approche in vivo en utilisant un
model animal de choc septique, une souris caractérisé par une hyperactivation de
caspase-1, nous avons pu observé une décroissance dans les formes longues de ces
enzymes dans le muscle du diaphragme. L'utilisation de souris défectueuses en caspase-l
vont nous démontrer si le clivage et la diminution dans la forme longue de ces enzymes
est réellement le résultat d'un clivage par caspase-1. Cela fournira un lien direct
entre l'activation de caspase-1 et l'inhibition de la glycolyse qui pourra avoir des
percussions importantes sur la perte de contractilité du muscle dans le choc septique.
111
CONTRIBUTION OF AUTHORS
The research conducted and presented in this thesis is entirely my own except that septic
shock rhice induced with LPS was done by Dr. Sabah Hussain's lab. Dr. Maya Saleh
supervised the majority of the work. This thesis was written by me with correction by Dr.
Maya Saleh.
iv
CONTRIBUTION OF AUTHORS
The research conducted and presented in this thesis is entirely my own except that septic
shock rtlice induced with LPS was done by Dr. Sabah Hussain's labo Dr. Maya Saleh
supervised the majority of the work. This thesis was written by me with correction by Dr.
Maya Saleh.
IV
ACKNOWLEDGEMENTS
I would like to acknowledge my supervisor, Dr. Maya Saleh, for giving me the
opportunity to do this project, as well as her supervision on my project.
I would like to thank all past and present members of Maya's lab, especially Amal Nadiri
and David Kalant for their help in my project. I would like to thank all the lab members
for all sorts of support.
I would like to thank my parents, sister and brother for their unconditional love. I want to
give my special thanks to my husband and my son, who have brought me so much love in
my life. Their love encourages me to cope with all the pressure I would encounter in my
life.
v
ACKNOWLEDGEMENTS
l would like to acknowledge my supervisor, Dr. Maya SaI eh, for gIVmg me the
opportunity to do this project, as weU as her supervision on my project.
l would like to thank aH past and present members of Maya's lab, especiaHy Amal Nadiri
and David Kalant for their help in my project. l would like to thank aH the lab members
for aU sorts of support.
l would like to thank my parents, sister and brother for their unconditionallove. l want to
give my special thanks to my husband and my son, who have brought me so much love in
my life. Their love encourages me to cope with aH the pressure l would encounter in my
life.
v
/~\
TABLE OF CONTENTS
ABSTRACT ii
RESUME iii
CONTRIBUTION OF AUTHORS iv
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vi
LIST OF FIGURES viii
LIST OF TABLES ix
ABREVIATIONS x
LITERATURE REVIEW: 1
Apoptosis 1
Caspase family 1
The general structure of caspases 2
Caspase activation 2
The extrinsic pathway 3
The intrinsic pathway 4
Granzyme B-initiated caspase-activation pathway 5
Caspase activation networks 5
Regulation of caspases in apoptosis 6
The Bcl-2 family 6
The IAP family 6
Caspase proteolytic properties 7
Caspase substrates in apoptosis 8
Caspase substrates involved in neuropathologies 8
Synthetic inhibitors 9
Naturally occurring protein inhibitors 9
Non-apoptotic functions of caspases 10
Caspase-1 11
Caspase-1 activation, the inflammasome and the pyroptosome 11
Caspase-1 function 12
Negative regulation of caspase-1 by caspase-12 14
Caspase-1 substrates 15
GOAL OF THIS STUDY.... 16
MATERIALS AND METHODS 17
vi
TABLE OF CONTENTS
ABSTRACT ................................................................................................................... ii , ,
RESUME ...................................................................................................................... iii
CONTRIBUTION OF AUTHORS .............................................................................. iv
ACKNOWLEDGEMENTS .......................................................................................... v
TABLE OF CONTENTS ............................................................................................. vi
LIST OF FIGURES .................................................................................................... viii
LIST OF TABLES ....................................................................................................... ix
ABREVIATIONS .......................................................................................................... x
LITERATURE REVIEW: ............................................................................................ 1
Apoptosis ..................................................................................................................... 1
Caspase fami1y ............................................................................................................. 1
The genera1 structure of caspases ................................................................................. 2
Caspase activation ........................................................................................................ 2
The extrinsic pathway ............................................................................................... 3
The intrinsic pathway ............................................................................................... 4
Granzyme B-initiated caspase-activation pathway .................................................... 5
Caspase activation networks ..................................................................................... 5
Regulation of caspases in apoptosis .............................................................................. 6
The Bcl-2 fami1y ...................................................................................................... 6
The IAP family ......................................................................................................... 6
Caspase proteolytic properties ...................................................................................... 7
Caspase substrates in apoptosis .................................................................................... 8
Caspase substrates involved in neuropathologies ...................................................... 8
Synthetic inhibitors ................................................................................................... 9
Naturally occurring protein inhibitors ....................................................................... 9
Non-apoptotic functions of caspases .......................................................................... 10
Caspase-1 ................................................................................................................... Il
Caspase-1 activation, the inflammasome and the pyroptosome ............................... Il
Caspase-1 function ................................................................................................. 12
Negative regulation of caspase-1 by caspase-12 ...................................................... 14
Caspase-1 substrates ............................................................................................... 15
GOAL OF THIS STUDY ............................................................................................ 16
MATERIALS AND METHODS ................................................................................ 17
VI
Cell culture 17
Human PBMC isolation and proliferation 17
Diagonal gel 17
In vitro cleavage assay with whole cell lysates 18
Western blot 18
RN A extraction and RT-PCR 19
Constructs and site-directed mutagenesis 19
In vitro cleavage of 35S methionine-labeled substrates 20
Caspase-1 activation with ATP/nigericin treatment 20
THP-1 cells Salmonella infection 21
Bacterial preparation for infection 21
Cell culture and infection 21
FLICAcasp"1 activity measurement 22
LDH measurement of THP-1 cells infected with Salmonella 22
Mice model of LPS-induced septic shock 22
RESULTS AND DISCUSION 23
1. Caspases-1 and -3 substrates identified by the diagonal gel approach 23
2. Caspase-1 targets in the glycolytic pathway 25
2.1 validation of the cleavage of the glycolysis enzymes by caspase-1 27
2.1.1 Validation of the caspase-1 cleaving glycolysis in vitro by western blot 27
2.1.2 Caspase-1 cleavage of in vitro transcribed and translated glycolysis enzymes 28
2.3 In vivo studies on the cleavage of the glycolysis enzymes upon activation of
Caspase-1 31
2.3.1 Activation of caspase-1 by transfection of procaspase-1 into HEK293 cells ...31
2.3.2 Activation of caspase-1 in THP-1 cells treated with LPS plus ATP or Nigericin
32
2.3.3 Activation of caspase-1 in Salmonella infected THP-1 cells 32
2.3.4 Examination of the glycolysis enzymes cleavage in the mice muscle in Septic
shock 34
2.3.5 The Glycolytic rate is reduced during Salmonella infection of THP-1 cells ....35
CONCLUSIONS AND FUTURE DIRECTIONS: 37
REFERENCE 82
APPENDIX I Caspase-1 substrate: G ATA-4 91
APPENDIX II 97
vn
Cell culture ................................................................................................................ 17
Human PB MC isolation and proliferation .................................................................. 17
Diagonal geL .............................................................................................................. 17
In vitro c1eavage assay with whole celllysates ........................................................... 18
Western blot. .............................................................................................................. 18
RNA extraction and RT-PCR ..................................................................................... 19
Constructs and site-directed mutagenesis ................................................................... 19
In vitro cleavage of 35S methionine-Iabeled substrates ................................................ 20
Caspase-l activation with ATP Inigericin treatment .................................................... 20
THP-l cells Salmonella infection ............................................................................... 21
Bacterial preparation for infection .......................................................................... 21
Cell culture and infection ........................................................................................ 21
FLICAcasp-l •• 22 actIvlty measurement. ............................................................................. .
LDH measurement ofTHP-l cells infected with Salmonella ...................................... 22
Mice model of LPS-induced septic shock ................................................................... 22
RESULTS AND DIS CU SION ..................................................................................... 23
1. Caspases-l and -3 substrates identified by the diagonal gel approach ..................... 23
2. Caspase-l targets in the glycolytic pathway ............................................................ 25
2.1 validation of the cleavage of the glycolysis enzymes by caspase-l ....................... 27
2.1.1 Validation of the caspase-l cleaving glycolysis in vitro by western blot. ........... 27
2.1.2 Caspase-l cleavage of in vitro transcribed and translated glycolysis enzymes 28
2.3 In vivo studies on the cleavage of the glycolysis enzymes upon activation of
Caspase-l ................................................................................................................... 31
2.3.1 Activation of caspase-l by transfection of procaspase-l into HEK293 cells ... 31
2.3.2 Activation of caspase-l in THP-l cells treated with LPS plus ATP or Nigericin
............................................................................................................................... 32
2.3.3 Activation of caspase-l in Salmonella infected THP-l cells ........................... 32
2.3.4 Examination of the glycolysis enzymes cleavage in the mice muscle in Septic
shock ......................................................................................................................... 34
2.3.5 The Glycolytic rate is reduced during Salmonella infection ofTHP-l cells .... 35
CONCLUSIONS AND FUTURE DIRECTIONS: ..................................................... 37
REFERENCE .............................................................................................................. 82
APPENDIX 1 Caspase-l substrate: G ATA-4 .......................................................... 91
APPEND IX II .............................................................................................................. 97
vu
r^-. LIST OF FIGURES
Figure 1 : Apoptotic Pathways 3
Figure 2 : Caspase Substrates Specificity 7
Figure 3 : Apoptosome vs. Inflammasome 12
Figure 4 : Schematic of Diagonal Gel Approach 42
Figure 5 : Map of Caspase-3 Digested Diagonal Gel 43
Figure 6 : Caspase-1 Substrate IL-1 ft Revealed by Western Blot in Diagonal Gel 44
Figure 7 : Diagonal Gel Map from Caspase-3 Digestion 45
Figure 8 : Pie Charts of Caspase-1 -3 Substrates 46
Figure 9 : Venn Diagram Comparing Caspase-1 and Caspase-3 Substtrates 47
Figure 10: Caspase-1 Targeted Substrates in Glycolytic Pathway 48
Figure 11: Western Blot of in vitro Cleavage of Glycolysis Enzymes by caspase-1 49
Figure 12: Western Blot of Comparing Glycolysis Enzymes Cleavage by Caspase-1 and -3 51
Figure 13: Comparision of ITT Glycolysis Enzymes Cleavage by Caspase-1 and -3 53
Figure 14: Calibrating Cleavage of ITT Glycolysis Enzymes by Caspase-1 55
Figure 15: Identification of Caspase-1 Cleavage Site in GAPDH 58
Figure 16: Identification of Caspase-1 Cleavage Site in a -Enolase 60
Figure 17: Western blot of Glycolysis Enzymes in ATP/Nigericin Treated THP-1 Cells. 62
Figure 18: Depletion of Glycolysis Enzymes in Salmonella infected THP-1 Cells 64
Figure 19: Depletion of Glycolysis Enzymes in Mice Muscle with Septic Shock 66
Figure 20: Change of Glycolysis rate in THP-1 cells after Salmonella infection 68
r~\
Vll l
LIST OF FIGURES
Figure 1 : Apoptotic Pathways ......................................................................................... 3
Figure 2 : Caspase Substrates Specificity ......................................................................... 7
Figure 3 : Apoptosome vs. Inflammasome ..................................................................... 12
Figure 4: Schematic of Diagonal Gel Approach ............................................................ 42
Figure 5: Map ofCaspase-3 Digested Diagonal Gel.. .................................................... 43
Figure 6 : Caspase-1 Substrate IL-lB Revealed by Western Blot in Diagonal Gel ...... .44
Figure 7 : Diagonal Gel Map from Caspase-3 Digestion ................................................ 45
Figure 8 : Pie Charts ofCaspase-1 -3 Substrates ........................................................... .46
Figure 9: Venn Diagram Comparing Caspase-1 and Caspase-3 Substtrates ................... 47
Figure 10: Caspase-1 Targeted Substrates in Glycolytic Pathway .................................. 48
Figure Il: Western Blot of in vitro Cleavage of Glycolysis Enzymes by caspase-1 ....... 49
Figure 12: Western Blot ofComparing Glycolysis Enzymes Cleavage by Caspase-1 and -3 51
Figure 13: Comparision ofITT Glycolysis Enzymes Cleavage by Caspase-1 and -3 ..... 53
Figure 14: Calibrating Cleavage ofITT Glycolysis Enzymes by Caspase-1 ................. 55
Figure 15: Identification of Caspase-1Cleavage Site in GAPDH .................................. 58
Figure 16: Identification of Caspase-1Cleavage Site in a -Enolase ............................. 60
Figure 17: Western blot of Glycolysis Enzymes in ATP/Nigericin Treated THP-1Cells. 62
Figure 18: Depletion of Glycolysis Enzymes in Salmonella infected THP-1 Cells ......... 64
Figure 19: Depletion of Glycolysis Enzymes in Mice Muscle with Septic Shock ......... 66
Figure 20: Change of Glycolysis rate in THP-1 cells after Salmonella infection ............ 68
V111
f~\ LIST OF TABLES
Table 1 Caspase-1 and -3 substrates obtained by diagonal gel approach 69
Table 2 Cloning primers for PCR 79
Table 3 Primers for potential cleavage sites mutagenesis 80
I
I
I
i
ix
LIST OF TABLES
Table 1 Caspase-l and -3 substrates obtained by diagonal gel approach ......................... 69
Table 2 Cloning primers for PCR .................................................................................. 79
Table 3 Primers for potential cleavage sites mutagenesis ............................................... 80
IX
ABREVIATIONS
APAF-1
APP
ASC
ATCC
ATP
BOC
cAMP
CARD
CrmA
CTL
DD
DED
DIAP
DISC
DR
DTT
EPO
FACs
FADD
FBS
FLICA
FMK
GAPDH
GLUT1
HIF
apoptosis protease-activating factor-1
amyloid (3 precursor protein
apoptosis-associated speckle-like containing protein
American type culture collection
adenosine triphosphate
benzyloxycarbonyl
cyclic adenosine monophosphate
caspase recruitment domain
cytokine response modifier A
cytotoxic T lymphocyte
death domain
death effector domain
Drosophila inhibitor of caspase
death-inducing signaling complex
death receptor
dithiothreitol
erythropoitin
fluorescent-activated cell sorting
Fas-associated protein with a death domain
fatal bovine serum
fluorochrome inhibitor of caspases
fluorometyl ketone
glyceraldehydes-3 -phosphate dehydroenase
glucose transporter 1
hypoxia-inducible factor
ABREVIATIONS
APAF-I
APP
ASC
ATCC
ATP
BOC
cAMP
CARD
CrmA
CTL
DD
DED
DIAP
DISC
DR
DTT
EPO
FACs
FADD
FBS
FUCA
FMK
GAPDH
GLUTI
HIF
apoptosis protease-activating factor-l
amy loid j3 precursor protein
apoptosis-associated speckle-like containing protein
American type culture collection
adenosine triphosphate
benzy loxycarbony 1
cyclic adenosine monophosphate
caspase recruitment domain
cytokine response modifier A
cytotoxic T lymphocyte
death domain
death effector domain
Drosophila inhibitor of caspase
death-inducing signaling complex
death receptor
dithiothreitol
erythropoitin
fluorescent-activated cell sorting
Fas-associated prote in with a death domain
fatal bovine serum
fluorochrome inhibitor of caspases
fluorometyl ketone
glyceraldehydes-3-phosphate dehydroenase
glucose transporter 1
hypoxia-inducible factor
x
HSP
IKB
IAP
ICAD
IL
IPAF
ITT
LB
LC-MS
LDH
LPS
LRR
MAPK
MBP-1
moi
MOMP
NAIP
NF-KB
PAGE
PARP
PBMC
PBS
PMA
PYD
Rb
RBC
RIP
heat shock protein
Inhibitor of NF-KB
inhibitor of apoptosis protein
inhibitor of caspase-activated Dnase
interleukin
ICE protease-activating factor
in vitro transcription and translation
Luria-Bertani
liquid chromatography-fed mass spectrometry
lactate dehydrogenase
lipopolysaccharide
Leucine rich repeats
mitogen-activated protein kinase
Myc promoter-binding protein-1
multiplicity of infection
mitochondrial outer membrane permeablilization
neuronal apoptosis inhibitor protein
nuclear Factor kappa B
polyacrylamide gel electrophoresis
poly (ADP-ribose) polymerase
peripheral blood mononuclear cells
phosphate buffered saline
phorbol-12-myristate-13 -acetate
Pyrin domain
retinoblastoma-associatedproteein
red blood cells
receptor interacting protein
r" HSP heat shock protein
IKB Inhibitor ofNF-KB
IAP inhibitor of apoptosis protein
ICAD inhibitor of caspase-activated Dnase
IL interleukin
IPAF ICE protease-activating factor
ITT in vitro transcription and translation
LB L uria-Bertani
LC-MS liquid chromatography-fed mass spectrometry
LDH lactate dehydrogenase
LPS lipopolysaccharide
LRR Leucine rich repeats
MAPK mitogen-activated prote in kinase
MBP-I Mye promoter-binding protein-l
mm multiplicity of infection
MOMP mitochondrial outer membtane permeablilization
NAIP neuronal apoptosis inhibitor protein
NF-KB nuclear Factor kappa B
PAGE polyacrylamide gel electrophoresis
PARP poly (ADP-ribose) polymerase
PBMC peripheral blood mononuclear cells
PBS phosphate buffered saline
PMA phorbol-12-myristate-13-acetate
PYD Pyrin domain
Rb retinoblastoma-associated proteein
RBC red blood cells
~ RIP receptor interacting prote in
Xl
/"~N SDS Sodium dodecyl sulfate
SREBP sterol regulatory element binding proteins
TBS tris-buffered saline
TIM triosephosphate isomerase
TNF tumor necrosis factor
TNF-R1 TNF receptor 1
TRADD TNFR-associated death domain
TRAF1 TNF receptor-associated factor-1
TRAIL TNF-related apoptosis-inducing ligand
XIAP X-chromasome-linked inhibitor of apoptosis protein
r^-.
Xll
(' SDS
SREBP
TBS
TIM
TNF
TNF-RI
TRADD
TRAFI
TRAIL
XIAP
Sodium dodecyl sulfate
sterol regulatory element binding proteins
tris-buffered saline
triosephosphate isomerase
tumor necrosis factor
TNF receptor 1
TNFR-associated death domain
TNF receptor-associated factor-l
TNF -related apoptosis-inducing ligand
X-chromasome-linked inhibitor of apoptosis protein
Xll
f~s LITERATURE REVIEW:
Apoptosis
Apoptosis is a genetically programmed cell death. This process can be triggered by a
variety of stimuli, including cytokines, hormones, viruses, and toxic insults (Creagh et al.,
2003). It is a fundamentally important process necessary for development and
homeostasis of multicellular organisms. Inappropriate apoptosis causes human diseases,
including neurodegenerative diseases, autoimmune disorders and cancer (Nicholson and
Thornberry, 1997). Apoptosis is the major form of cell death, which is characterized by
cell shrinkage, chromatin condensation, DNA fragmentation and membrane blebbing, all
of which result in recognition and phagocytosis of apoptotic cells by phagocytes, thereby
preventing an inflammatory response. This process is coordinated by a group of proteases,
the caspases (Nicholson and Thornberry, 1997).
Caspase family
The name caspase (derived from cysteinyl-aspartate-specific proteinase) illustrates two
distinguishing features of these enzymes: first, their catalysis is governed by a critical
conserved cysteine residue; second, they specifically cleave after an Aspartate amino acid.
To date, 14 mammalian family members have been identified. (Caspase-15 has been
recently identified as an apoptotic caspase expressed in various mammalian species but
not in humans (Eckhart et al., 2005)). 13 human caspases are known. A phylogenetic
analysis classifies the family into two subfamilies: the caspase-1 subfamily (caspases-1,
-4, -5, and -12) and the caspase-3 subfamily (caspases-2, 3, 6, 7, 8, 9, and 10).
Caspase-14 is confined to the skin where it acts in skin differentiation. Caspase-1
subfamily members predominantly play a role in inflammation, while caspase-3
subfamily members are mainly involved in apoptosis (Nicholson, 1999).
f~\
1
LITERATURE REVIEW:
Apoptosis
Apoptosis is a genetically programmed cell death. This process can be triggered by a
variety of stimuli, inc1uding cytokines, hormones, viroses, and toxic insults (Creagh et al.,
2003). It is a fundamentally important process necessary for development and
homeostasis of multicellular organisms. Inappropriate apoptosis causes human diseases,
inc1uding neurodegenerative diseases, auto immune disorders and cancer (Nicholson and
Thomberry, 1997). Apoptosis is the major form of cell death, which is characterized by
cell shrinkage, chromatin condensation, DNA fragmentation and membrane blebbing, all
of which result in recognition and phagocytosis of apoptotic cells by phagocytes, thereby
preventing an inflammatory response. This process is coordinated by a group of proteases,
the caspases (Nicholson and Thomberry, 1997).
Caspase family
The name caspase (derived from 0'steinyl-aspartate-specific proteinase) illustrates two
distinguishing features of these enzymes: first, their catalysis is govemed by a critical
conserved cysteine residue; second, they specifica1ly c1eave after an Aspartate amino acid.
To date, 14 mammalian family members have been identified. (Caspase-15 has been
recently identified as an apoptotic caspase expressed in various mammalian species but
not in humans (Eckhart et al., 2005)). 13 human caspases are known. A phylogenetic
analysis classifies the family into two subfamilies: the caspase-l subfamily (caspases-l,
-4, -5, and -12) and the caspase-3 subfamily (caspases-2, 3, 6, 7, 8, 9, and 10).
Caspase-14 is confined to the skin where it acts in skin differentiation. Caspase-l
subfamily members predominantly play a role in inflammation, while caspase-3
subfamily members are mainly involved in apoptosis (Nicholson, 1999).
1
The general structure ofcaspases
Caspases are synthesized as catalytically dormant proenzymes containing three domains:
an N-terminal prodomain, a large subunit and a small subunit. They all have a conserved
QACXG sequence in their catalytic center. In general, caspases can autocleave
themselves or cleave other caspases. Based on their known or hypothetical roles in
apoptosis, caspases are further divided into two functional subgroups: initiator and
executioner caspases. Initiator caspases (caspases-1, 2, 8, 9 and 10) are responsible for
initiating caspase-activation cascades. The initiator caspases have long prodomains
containing caspase recruitment domains (CARDs) or death effector domains (DEDs),
which promote caspase activation. The downstream or executioner caspases (caspases-3,
6, 7, and as we present in this thesis caspase-1) are responsible for the actual dismantling
of the cell during apoptosis and pyroptosis, which is a caspase-1-induced cell death
associated with inflammation. Executioner caspases usually have short prodomains.
Caspase activation
The initiator caspases exist as monomers in the cytosol, and are activated by
oligomerization in macromolecular complexes assembled by scaffolding molecules.
Oligomerization of initiator caspases is triggered by the assembly of platforms that recruit
caspases into close proximity. When oligomerized within macromolecular structures such
as the DISC (death-inducing signaling complex), the apoptosome or the inflammasome,
initiator caspases form an active site and subsequent cleavage within the inter-domain
linker stabilizes their oligomers (Boatright et al., 2003, Green, 2003 #25; Green, 2003).
The executioner caspases exist as inactive proform dimers. Their activation occurs
through cleavage by the initiator caspases (Green, 2003). The activation pathways of the
apoptotic caspases are discussed below as the extrinsic, intrinsic and granzyme B
activation pathways (Creagh et al., 2003). The activation of the inflammatory caspases is
reviewed in the caspase-1 section.
2
The general structure of caspases
Caspases are synthesized as catalytically dormant proenzymes containing three domains:
an N-terminal prodomain, a large subunit and a small subunit. They aIl have a conserved
QACXG sequence in their catalytic center. In general, caspases can autocleave
themselves or cleave other caspases. Based on their known or hypothetical roles in
apoptosis, caspases are further divided into two functional subgroups: initiator and
executioner caspases. Initiator caspases (caspases-l, 2, 8, 9 and 10) are responsible for
initiating caspase-activation cascades. The initiator caspases have long prodomains
containing caspase recruitment domains (CARDs) or death effector domains (DEDs),
which promote caspase activation. The downstream or executioner caspases (caspases-3,
6, 7, and as we present in this thesis caspase-l) are responsible for the actual dismantling
of the cell during apoptosis and pyroptosis, which is a caspase-l-induced cell death
associated with inflammation. Executioner caspases usually have short prodomains.
Caspase activation
The initiator caspases exist as monomers in the cytosol, and are activated by
oligomerization in macromolecular complexes assembled by scaffolding molecules.
Oligomerization of initiator caspases is triggered by the assembly of platforms that recruit
caspases into close proximity. When oligomerized within macromolecular structures such
as the DISC (death-inducing signaling complex), the apoptosome or the inflammasome,
initiator caspases form an active site and subsequent cleavage within the inter-domain
linker stabilizes their oligomers (Boatright et al., 2003, Green, 2003 #25; Green, 2003).
The executioner caspases exist as inactive proform dimers. Their activation occurs
through cleavage by the initiator caspases (Green, 2003). The activation pathways of the
apoptotic caspases are discussed below as the extrinsic, intrinsic and granzyme B
activation pathways (Creagh et al., 2003). The activation of the inflammatory caspases is
reviewed in the caspase-l section.
2
The extrinsic pathway
This pathway is responsible for eliminating activated T cells (AICD-activation-induced
cell death), as well as virally infected or transformed cells. The 3 molecular players that
are engaged at the Extrinsic
death-inducing signaling
complex or DISC of the
extrinsic pathways are: 1)
Death receptors, 2) FADD, and
3) the initiator caspases -
caspases-8 or -10. Death
receptors include the TNF
receptor 1 (TNF-R1), Fas also
known as CD95, and the
TRAIL receptors DR4 and
DR5. FADD, also known as
FAS-associated protein with a
death domain, is a bimodular
adaptor that contains a death
domain DD but also a death
effector domain DED. DDs,
DEDs, CARDs (caspase
recruitment domains) and pyrin
domains PyDs belong to a
group of structural domains
Granzyme B
C*spas»-7
Figure 1 Apoptosis Pathways (Creagh, Conroy et al. 2003)
known as the "death fold" domains that consist of 6 cc-helices and that mediate
3
The extrinsic pathway
This pathway is responsible for eliminating activated T cells (AICD-activation-induced
cell death), as well as virally infected or transformed cells. The 3 molecular players that
are engaged at the
death-inducing signaling
complex or DISC of the
extrinsic pathways are: 1)
Death receptors, 2) FADD, and
3) the initiator caspases -
caspases-8 or -10. Death
receptors include the TNF
receptor 1 (TNF-Rl), Fas also
known as CD95, and the
TRAIL receptors DR4 and
DR5. FADD, also known as
F AS-associated protein with a
death domain, is a bimodular
adaptor that contains a death
domain DD but also a death
effector domain DED. DDs,
DEDs, CARDs (caspase
recruitment domains) and pyrin
domains PyDs belong to a
group of structural domains
Extrinsic
GranzymeB
FADO lntrinsic
Figure 1 Apoptosis Pathways (Creagh, Conroy et al. 2003)
known as the "death fold" domains that consist of 6 a-helices and that mediate
3
homotypic protein-protein interactions (e.g. DD-DD, DED-DED, CARD-CARD and
PyD-PyD). Upon binding to their ligands, death receptors oligomerize on the cell surface
and assemble the DISC. In their cytoplasmic tails, death receptors contain a death domain
(DD). In response to ligand binding, the death receptor DD associates with the death
domain of FADD, which in turn recruits Caspase-8 via its death effector domain (DED).
This results in the activation of the initiator caspases-8 and 10. The activated initiator
caspases can then cleave and activate executioner caspases (figure 1) (Earnshaw et al.,
1999). TNFR1 binds to an additional adapter protein TRADD, which in turn binds to
FADD. New evidence shows that the activation via TNF-R1 may happen in two steps:
TNF-R1 associates with TRADD upon binding to its ligand, and then dissociates from
TRADD due to TRADD undergoing some modification, possibly phosphorylation, which
allows TRADD to recruit and activate caspase-8 within the cytosol (Micheau and
Tschopp, 2003).
The intrinsic pathway
This pathway is responsible for the maintenance of normal cell number in neuronal
development as well as apoptosis in response to environmental insults. Activation of this
pathway is initiated by stimuli such as cytotoxic drugs, heat shock, ionizing radiation and
other cellular stresses leading to mitochondrial outer membrane permeabilization
(MOMP). MOMP results in cytochrome c release from the mitochondrial intermembrane
space into the cytosol. Cytosolic cytochrome c binds to the WD40 repeat domain in
APAF-1 (apoptosis protease-activating factor-1), which induces an ATP-mediated
assembly of a heptamer complex and the recruitment of pro-caspase-9 via binding of the
caspase-9 CARD to the CARD in APAF-1. This protein complex is known as the
apoptosome. The activated caspase-9 then cleaves excutioner caspases (figure 1) (Creagh
et al., 2003).
4
homotypic protein-protein interactions (e.g. DD-DD, DED-DED, CARD-CARD and
PyD-PyD). Upon binding to their ligands, death receptors oligomerize on the cell surface
and assemble the DISC. In their cytoplasmic tails, death receptors contain a death domain
(DD). In response to ligand binding, the death receptor DD associates with the death
domain of FADD, which in turn recruits Caspase-8 via its death effector domain (DED).
This results in the activation of the initiator caspases-8 and 10. The activated initiator
caspases can then cleave and activate executioner caspases (figure 1) (Earnshaw et al.,
1999). TNFR1 binds to an additional adapter protein TRADD, which in turn binds to
FADD. New evidence shows that the activation via TNF-R1 may happen in two steps:
TNF-Rl associates with TRADD upon binding to its ligand, and then dissociates from
TRADD due to TRADD undergoing sorne modification, possibly phosphorylation, which
allows TRADD to recruit and activate caspase-8 within the cytosol (Micheau and
Tschopp, 2003).
The intrinsic pathway
This pathway is responsible for the maintenance of normal cell number in neuronal
development as weIl as apoptosis in response to environmental insults. Activation of this
pathway is initiated by stimuli such as cytotoxic drugs, heat shock, ionizing radiation and
other cellular stresses leading to mitochondrial outer membrane permeabilization
(MOMP). MOMP results in cytochrome c release from the mitochondrial intermembrane
space into the cytosol. Cytosolic cytochrome c binds to the WD40 repeat domain in
APAF -1 (apoptosis protease-activating factor-1), which induces an ATP-mediated
assembly of a heptamer complex and the recruitment of pro-caspase-9 via binding of the
caspase-9 CARD to the CARD in APAF-l. This protein complex is known as the
apoptosome. The activated caspase-9 then cleaves excutioner caspases (figure 1) (Creagh
et al., 2003).
4
I Granzyme B-initiated caspase-activation pathway
Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural
Killer cells, shares with caspases a preference for aspartate residues in the PI position of
its substrates. It is the only protease other than caspases known to cleave caspases
(Nicholson, 1999). It can cleave caspases-3 and -8 as well as the caspase-8 substrate Bid
and initiates apoptosis, building up an alternative apoptosis pathway in virally infected
and tumor cell (figure 1) (Atkinson et al , 1998; Medema et al , 1997)
Caspase activation networks
During apoptosis, initiator caspases are activated to cleave executioner caspases. Caspase
activation in the extrinsic pathway proceeds from the death receptor induced caspase-8
cleaving of caspase-3 which cleaves caspase-6; the intrinsic pathway proceeds from the
» cytochrome c:Apaf-l apoptosome complex by caspase-9 cleaving caspase-3 and
caspases-6 and -7. At the same time, downstream caspases can cleave upstream caspases.
For example, caspase-6 can cleave procaspases-8 and -10, and caspase-3 can cleave
procaspase-9. The cleavage of upstream caspases by downstream caspases acts as a
positive feedback mechanism to enhance apoptosis (Earnshaw et al., 1999).The protein
Bid serves as a "cross-talking" molecule that transmits the apoptotic signal from the
extrinsic to the intrinsic pathway. When caspase-8 is activated via the extrinsic pathway, i
, it cleaves Bid into its active form, tBid (truncated Bid). tBid translocates to the
mitochondria and activates pro-apoptotic members of the Bcl-2 family, Bax and Bak, to
) cause mitochondrial outer membrane permeabilization (MOMP). In addition, the
• mitochondrial proteins Smac/DIABLO and Omi/HtrA2 are released to prevent the
I inhibition of caspases mediated by the IAPs (inhibitors of apoptosis proteins). Granzyme
B also cleaves Bid into tBid and feeds the apoptotic signal to the intrinsic pathway
O (Green, 2003)
5
Granzyme B-initiated caspase-activation pathway
Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural
Killer cells, shares with caspases a preference for aspartate residues in the Pl position of
its substrates. l t is the only protease other than caspases known to cleave caspases
(Nicholson, 1999). It Can cleave caspases-3 and -8 as weIl as the caspase-8 substrate Bid
and initiates apoptosis, building up an alternative apoptosis pathway in virally infected
and tumor cell (figure 1) (Atkinson et al., 1998; Medema et al., 1997)
Caspase activation networks
During apoptosis, initiator caspases are activated to cleave executioner caspases. Caspase
activation in the extrinsic pathway proceeds from the death receptor induced caspase-8
cleaving of caspase-3 which cleaves caspase-6; the intrinsic pathway proceeds from the
cytochrome c:Apaf-1 apoptosome complex by caspase-9 cleaving caspase-3 and
caspases-6 and -7. At the same time, downstream caspases can cleave upstream caspases.
For example, caspase-6 Can cleave procaspases-8 and -10, and caspase-3 Can cleave
procaspase-9. The cleavage of upstream caspases by downstream caspases acts as a
positive feedback mechanism to enhance apoptosis (Earnshaw et al., 1999).The protein
Bid serves as a "cross-talking" molecule that transmits the apoptotic signal from the
extrinsic to the intrinsic pathway. When caspase-8 is activated via the extrinsic pathway,
it cleaves Bid into its active form, tBid (truncated Bid). tBid translocates to the
mitochondria and activates pro-apoptotic members of the Bcl-2 family, Bax and Bak, to
cause mitochondrial outer membrane permeabilization (MOMP). In addition, the
mitochondrial proteins Smac/DIABLO and OmiIHtrA2 are released to prevent the
inhibition of caspases mediated by the lAPs (inhibitors of apoptosis proteins). Granzyme
B also cleaves Bid into tBid and feeds the apoptotic signal to the intrinsic pathway
(Green, 2003)
5
Regulation ofcaspases in apoptosis
The activation of caspases is under the control of two protein families: 1) the pro- and
anti-apoptotic proteins of the Bcl-2 family, and 2) the IAP (inhibitor of apoptosis protein)
family.
The Bcl-2 family
The Bcl-2 family has anti- and pro-apoptotic members. During apoptosis, mitochondrial
outer membrane permeabilization (MOMP) occurs. Anti-apoptotic members of the Bcl-2
family (Bcl-2, Bcl-XL, Bcl-w and Mcl-1) prevent MOMP, and thus prevent cytochrome c
release and the consequent apoptosome assembly and caspase-9 activation. On the
contrary, the pro-apoptotic members BH123 proteins (Bax, Bak and Bok), when activated,
oppose the actions of the anti-apoptotic members and promote MOMP. The BH123
proteins are activated by another set of BH3-only proteins (Bim, Bid, Bad, Bmf, BNIP-3,
Puma, Noxa) (Green, 2003)
The IAP family
Bcl-2 family members regulate caspases by acting upstream of the mitochondria in the
intrinsic pathway. Downstream of the mitochondria, when procaspase-3 is cleaved by
caspase-9, although enzymatically active at this point, caspase-3 is under a "brake"
because it is bound by XIAP (X-chromosome-linked inhibitor of apoptosis protein), a
member of the IAP family. Relief of inhibition of caspase-3 by XIAP is facilitated by
competition of binding of Smac/DIABLO or Omi/Htra2 (released from mitochondria
during MOMP) to XIAP (Du et al., 2000; Verhagen et al., 2000).
6
Regulation of caspases in apoptosis
The activation of caspases is under the control of two protein families: 1) the pro- and
anti-apoptotic proteins of the Bel-2 family, and 2) the IAP (inhibitor of apoptosis protein)
family.
The Bcl-2 family
The Bel-2 family has anti- and pro-apoptotic members. During apoptosis, mitochondrial
outer membrane permeabilization (MOMP) occurs. Anti-apoptotic members of the Bel-2
family (Bel-2, Bel-XL, Bel-w and Mel-l) prevent MOMP, and thus prevent cytochrome c
release and the consequent apoptosome assembly and caspase-9 activation. On the
contrary, the pro-apoptotic members BH123 proteins (Bax, Bak and Bok), when activated,
oppose the actions of the anti-apoptotic members and promote MOMP. The BH123
proteins are activated by another set of BH3-only proteins (Bim, Bid, Bad, Bmf, BNIP-3,
Puma, Noxa) (Green, 2003)
The IAP family
Bcl-2 family members regulate caspases by acting upstream of the mitochondria in the
intrinsic pathway. Downstream of the mitochondria, when procaspase-3 is eleaved by
caspase-9, although enzymatically active at this point, caspase-3 is under a "brake"
because it is bound by XIAP (X-chromosome-linked inhibitor of apoptosis protein), a
member of the IAP family. Relief of inhibition of caspase-3 by XIAP is facilitated by
competition of binding of SmaclDIABLO or OmiIHtra2 (released from mitochondria
during MOMP) to XIAP (Du et al., 2000; Verhagen et al., 2000).
6
Caspase proteolytic properties
Caspases are cysteine proteases. They are distinct from other cysteine proteases owing to
their absolute requirement of an Asp in the PI position N-terminus of the scissile bond.
Of the currently known human and mouse proteases, only Grairzyme B shares this
specificity (Nicholson, 1999). The residue after the scissile bond (PI') is also important
in that charged or bulky residues are poorly tolerated; Gly, Ala, Thr, Ser and Asn allow
efficient catalysis, but Glu, Asp, Lys, Arg, Trp do not (Stennicke et al., 2000). Substrate
specificity is determined by the residue at the P4 position. Screening of a positional
scanning combinatorial substrate library divided caspases into three groups. Group I
caspases (1,4, and 5) prefer bulky hydrophobic amino acids such as Tyr or Trp in the P4
position. Group II caspases (2, 3, and 7) require a P4 Asp. Group III caspases (6, 8, 9,
and 10) prefer branched chain aliphatic amino acids in the P4 position (Nicholson, 1999)
(Figure 2).
jj^^gmbf&im&£j|gijgj Mjwuwm£>wgj||
Group)
Group li
Group III
Hydrophobic
Asp
Aliphatic
Glu V
Xxx Asp
Caspases-1,4, 5, 13
Caspases-2, 3, 7, CED-3
Caspases-6, 8,9. 10, (Granzyme B)
Figure 2 Caspase Substrates Specificity (Nicholson 1999)
For natural proteins, however, the presence of a preferred cleavage site consisting of four
specific amino acids is not sufficient. Enzyme regions distant from the catalytic site that
participate in substrate binding (exosites) have been implicated in the recognition of
7
Caspase proteolytic properties
Caspases are cysteine proteases. They are distinct from other cysteine proteases owing to
their absolute requirement of an Asp in the Pl position N-terminus of the scissile bond.
Of the currently known human and mouse proteases, only Granzyme B shares this
specificity (Nicholson, 1999). The residue after the scissile bond (P l ') is also important
in that charged or bulky residues are po orly tolerated; Gly, Ala, Thr, Ser and Asn allow
efficient catalysis, but Glu, Asp, Lys, Arg, Trp do not (Stennicke et al., 2000). Substrate
specificity is determined by the residue at the P4 position. Screening of a positional
scanning combinatorial substrate library divided caspases into three groups. Group 1
caspases (1, 4, and 5) prefer bulky hydrophobie amino acids such as Tyr or Trp in the P4
position. Group II caspases (2, 3, and 7) require a P4 Asp. Group III caspases (6, 8, 9,
andlO) pre fer branched chain aliphatic amino acids in the P4 position (Nicholson, 1999)
(Figure 2).
Group 1
Group Il
Group III
y
Hydrophobie ] V
Asp Glu Xxx Asp
A1iphatic
Caspases-1, 4, 5, 13
Caspases-2, 3, 7, CED·3
Caspases-6. 8. 9. 10, (Granzyme B)
Figure 2 Caspase Substrates Specificity (Nicholson 1999)
For natural proteins, however, the presence of a preferred c1eavage site consisting of four
specifie amino acids is not sufficient. Enzyme regions distant from the catalytic site that
participate in substrate binding (exosites) have been implicated in the recognition of
7
natural substrates. One example is derived from the crystal structure of caspase-8 bound
to its natural inhibitor p35, which is the only caspase structure solved with a natural
substrate bound to the caspase. p35 contains a loop that interacts with a face of caspase-8
distant from its active site, suggesting that exosites interactions with the substrate might
also be important (Fisher et al., 1999; Xu et al , 2001). The exosite interactions might
explain the discrepancies in the cleavage consensuses between synthetic and natural
substrates.
Caspase substrates in apoptosis
During apoptosis, caspases cleave a large number of proteins to disrupt cellular functions,
which accounts for the phenotype seen in cells undergoing apoptosis. Caspases cleave
key structural components of the cytoskeleton such as actin, fodrin, vimentin and keratin
etc., which contributes to cell shrinkage and cell detachment, as well as interrupts
antiapoptotic integrin signaling. Caspases cleave ICAD (inhibitor of caspase-activated
Dnase). Cleavage of ICAD releases CAD liberating it to translocate to the nucleus where
it fragments DNA. Caspases cleave numerous proteins involved in the transducation and
amplification of apoptotic signals. For example, caspases target the survival NF-KB
pathway, cleaving p65 and IKB-CX, TRAF-1 and RIP-1. Caspases also cleave critical
proteins involved in the regulation of the cell cycle, such as cyclin E, MDMX, Rb,
causing cell cycle attenuation. Additionally, caspases cleave many proteins involved in
the translation machinery, resulting in the blockade of protein translation (Fischer et al.,
2003).
Caspase substrates involved in neuropathologies
The cleavage of specific substrates is directly linked to pathogenesis of certain
neurodegenerative disorders. Huntingtin, which is mutated in Huntington's Disease and
8
,~ natural substrates. One example is derived from the crystal structure of caspase-8 bound
to its natural inhibitor p35, which is the only caspase structure solved with a natural
substrate bound to the caspase. p35 contains a loop that interacts with a face of caspase-8
distant from its active site, suggesting that exosites interactions with the substrate might
also be important (Fisher et al., 1999; Xu et al., 2001). The exosite interactions might
explain the discrepancies in the cleavage consensuses between synthetic and natural
substrates.
Caspase substrates in apoptosis
During apoptosis, caspases cleave a large number of proteins to disrupt cellular functions,
which accounts for the phenotype seen in cells undergoing apoptosis. Caspases cleave
key structural components of the cytoskeleton such as actin, fodrin, vimentin and keratin
etc., which contributes to cell shrinkage and cell detachment, as well as interrupts
antiapoptotic integrin signaling. Caspases cleave ICAD (inhibitor of caspase-activated
Dnase). Cleavage of ICAD releases CAD liberating it to translocate to the nucleus where
it fragments DNA. Caspases cleave numerous proteins involved in the transducation and
amplification of apoptotic signaIs. For example, caspases target the survival NF-KB
pathway, cleaving p65 and IKB-a, TRAF-l and RIP-l. Caspases also cleave critical
proteins involved in the regulation of the cell cycle, such as cyclin E, MDMX, Rb,
causing cell cycle attenuation. Additionally, caspases cleave many proteins involved in
the translation machinery, resulting in the blockade of protein translation (Fischer et al.,
2003).
Caspase substrates involved in neuropathologies
The cleavage of specifie substrates is directly linked to pathogenesis of certain
neurodegenerative disorders. Huntingtin, which is mutated in Huntington' s Disease and
8
possesses an expanded polyglutamine stretch in its N-terminus, is cleaved by caspase-3,
and it is the aggregation of its N-terminal fragments in neurons which is suggested to
cause the disease (Zhang et al., 2003b). APP, the amyloid |3 precursor protein involved in
Alzheimer's disease, is additionally cleaved by caspase-3 at VEVD664. The consequent
generation of the cytosolic fragment (C31) is associated with cell toxicity. Transgenic
mice expressing an uncleavable mutant of APP (mutation of the cleavage site to alanine
VEVD6641 A) are protected from the disease (Galvan et al., 2006).
Synthetic inhibitors
The sufficiency of a P4-P1 four amino acid recognition motif for caspases serves as the
basis of inhibitor design. Reversible inhibitors of caspases are generated by coupling
caspase-specific peptides to certain aldehyde, nitrile or ketone compounds; irreversible
inhibitors are generated by coupling caspase-specific peptides to fluoromethyl ketone
(FMK). To enhance cellular permeability, the inhibitors are synthesized with a
benzyloxycarbonyl group (also known as BOC or Z), and they are widely used in both in
vitro cell culture and in vivo studies. Examples of commercially available inhibitors are
z-YVAD-FMK for caspase-1; z-DEVD-FMK for caspase-3; z-IETD-FMK for caspase-8
and a pan-caspase inhibitor z-VAD-FMK (Nicholson, 1999).
Naturally occurring protein inhibitors
Cytokine response modifier A (CrmA) is a 38 KDa protein from cowpox virus, which
facilitates viral infection through inhibition of cytokine production and apoptosis by
efficient inhibition of both caspases-1 and -8 (Komiyama et al., 1994). p35 is another
viral protein from baculovirus. Cleavage of p35 by a caspase results in the formation of a
caspase-p35 complex. The presence of this complex prevents caspases from initiating the
9
possesses an expanded polyglutamine stretch in its N-terminus, is cleaved by caspase-3,
and it is the aggregation of its N-terminal fragments in neurons which is suggested to
cause the disease (Zhang et al., 2003b). APP, the amyloid j3 precursor protein involved in
Alzheimer's disease, is additionally cleaved by caspase-3 at VEVD664. The consequent
generation of the cytosolic fragment (C31) is associated with cell toxicity. Transgenic
mice expressing an uncleavable mutant of APP (mutation of the cleavage site to alanine
VEVD664 + A) are protected from the disease (Galvan et al., 2006).
Synthetic inhibitors
The sufficiency of a P4-P1 four amino acid recognition motif for caspases serves as the
basis of inhibitor design. Reversible inhibitors of caspases are generated by coupling
caspase-specific peptides to certain aldehyde, nitrile or ketone compounds; irreversible
inhibitors are generated by coupling caspase-specific peptides to fluoromethyl ketone
(FMK). To enhance cellular permeability, the inhibitors are synthesized with a
benzyloxycarbonyl group (also known as BOC or Z), and they are widely used in both in
vitro cell culture and in vivo studies. Examples of commercially available inhibitors are
z-YVAD-FMK for caspase-1; z-DEVD-FMK for caspase-3; z-IETD-FMK for caspase-8
and a pan-caspase inhibitor z-VAD-FMK (Nicholson, 1999).
Naturally occurring protein inhibitors
Cytokine response modifier A (CrmA) is a 38 KDa protein from cowpox virus, which
facilitates viral infection through inhibition of cytokine production and apoptosis by
efficient inhibition of both caspases-1 and -8 (Komiyama et al., 1994). p35 is another
viral protein from baculovirus. Cleavage of p35 by a caspase results in the formation of a
caspase-p35 complex. The presence ofthis complex prevents caspases from initiating the
9
apoptotic cascade (Xue and Horvitz, 1995).
Non-apoptotic functions ofcaspases
Caspases play an essential role in apoptosis. But recent studies have uncovered novel
functions of caspases in non-apoptotic processes including the inflammatory response,
immune cell proliferation, cell differentiation and cell migration. The major function of
caspase-1 is in regulation of the inflammatory response by processing pro-IL-lfS,
pro-IL-18 and pro-IL-33 into their mature cytokine forms (Li et al., 1995; Schmitz et al.,
2005). New evidence show that caspase-1 could also contribute to inflammation by the
activation of the NF-KB pathway through the interaction between the CARD domain of
caspase-1 and the kinase RIP1 (Lamkanfi et al., 2004). Caspase-3, the main death
effector caspase, can process pro-IL-16 into its active form (Zhang et al., 1998),
suggesting a role of caspase-3 in inflammation. Caspase-8 appears to play an essential
role in T-cell proliferation and activation. It has been shown that caspase-8 cleaves the
kinase Weel during T-cell proliferation (Alam et al., 1999), which prevents
phosphorylation of the cell cycle-regulating kinase Cdc2 needed to promote cell cycle
progression. Caspase-8 is also implicated in cell differentiation. Conditional knockout of
caspase-8 in the T cell-lineage revealed a defect in T cell homeostasis and activation in
the absence of caspase-8 (Salmena and Hakem, 2005). Additionally, conditional
knock-out of caspase-8 in the myelomonocytic lineage resulted in an arrest in
macrophage differentiation (Kang et al., 2004). Caspases are also involved in the
differentiation process associated with enucleation of erythrocytes and keratinocytes
(Zandy et al., 2005; Zermati et al., 2001). The enucleation process exhibits some
similarities to apoptosis such as chromatin condensation and degradation of nuclear
components (Morioka et al., 1998). Caspases are probably activated through a
mitochondrial-dependent pathway and cleave proteins such as lamin and actin, which are
responsible for nuclear disassembly and chromatin condensation (Zermati et al., 2001).
10
apoptotic cascade (Xue and Horvitz, 1995).
Non-apoptotie lunetions 01 easpases
Caspases play an essential role in apoptosis. But recent studies have uncovered novel
functions of caspases in non-apoptotic processes including the inflammatory response,
immune cell proliferation, cell differentiation and cell migration. The major function of
caspase-1 is in regulation of the inflammatory response by processing pro-IL-1~,
pro-IL-18 and pro-IL-33 into their mature cytokine forms (Li et al., 1995; Schmitz et al.,
2005). New evidence show that caspase-1 could also contribute to inflammation by the
activation of the NF -KB pathway through the interaction between the CARD domain of
caspase-1 and the kinase RIP1 (Lamkanfi et al., 2004). Caspase-3, the main death
effector caspase, can process pro-IL-16 into its active form (Zhang et al., 1998),
suggesting a role of caspase-3 in inflammation. Caspase-8 appears to play an essential
role in T-cell proliferation and activation. It has been shown that caspase-8 cleaves the
kinase Wee1 during T-cell proliferation (Alam et al., 1999), which prevents
phosphorylation of the cell cycle-regulating kinase Cdc2 needed to promote cell cycle
progression. Caspase-8 is also implicated in cell differentiation. Conditional knockout of
caspase-8 in the T cell-lineage revealed a defect in T cell homeostasis and activation in
the absence of caspase-8 (Salmena and Hakem, 2005). Additionally, conditional
knock-out of caspase-8 in the myelomonocytic lineage resulted in an arrest in
macrophage differentiation (Kang et al., 2004). Caspases are also involved in the
differentiation process associated with enucleation of erythrocytes and keratinocytes
(Zandy et al., 2005; Zermati et al., 2001). The enucleation process exhibits sorne
similarities to apoptosis such as chromatin condensation and degradation of nuclear
components (Morioka et al., 1998). Caspases are probably activated through a
mitochondrial-dependent pathway and cleave proteins such as lamin and actin, which are
responsible for nuclear disassembly and chromatin condensation (Zermati et al., 2001).
10
Although caspases are activated in those cell types during enuleation, the activation level
is not sufficient to dismantle the cell, and therefore only results in more selective
targeting of substrates.
Caspase-1
Caspase-l is best known for its role in inflammation, where it cleaves pro-IL-ip and
pro-IL18 into their active cytokine forms (Li et al., 1995). It is also implicated in cell
death. A novel form of cell death induced by invasive bacteria, the pyroptosis, has been
proposed and this form of cell death is dependent on caspase-1 (Fink and Cookson,
2005).
Caspase-1 activation, the inflammasome and the pyroptosome
Structurally, caspase-1 has a long prodomain and is predicted to act as an initiator
caspase. Like other initiator caspases, its activation is triggered by the assembly of a
multimeric protein complex. This complex is known as the inflammasome, which
resembles structurally the apoptosome. In this complex, NLR (NACHT-LRR) family
members such as NALP1, 3, and IPAF serve as scaffolding proteins (Martinon and
Tschopp, 2005). The activation of caspase-1 in the inflammasome is comparable to that
of caspase-9 in the apoptosome (Figure 3). In general, NLR members are typically
composed of three domains: a Leucine Rich Repeat (LRR) ligand sensing domain, a
NACHT nucleotide binding and oligomerization domain, and a CARD or Pyrin domain
(PYD) for caspase recruitment (Martinon and Tschopp, 2004; Nadiri et al., 2006).
Four types of inflammasomes have been characterized and shown to activate caspase-1:
NALP-1, NALP-3, NAIP5 and IPAF. (Martinon and Tschopp, 2004; Molofsky et al.,
2006). The interaction between NALP proteins and caspase-1 is not direct but depends on
the adaptor ASC; whereas, IPAF can activate caspase-1 directly via its CARD
11
1
•
Although caspases are activated in those cell types during enuleation, the activation level
is not sufficient to dismantle the cell, and therefore only results in more selective
targeting of substrates.
Caspase-l
Caspase-l is best known for its role in inflammation, where it cleaves pro-IL-l (3 and
pro-ILl8 into their active cytokine forms (Li et al., 1995). It is also implicated in cell
death. A novel form of cell death induced by invasive bacteria, the pyroptosis, has been
proposed and this form of cell death is dependent on caspase-l (Fink and Cookson,
2005).
Caspase-l activation, the inflammasome and the pyroptosome
Structurally, caspase-l has a long prodomain and is predicted to act as an initiator
caspase. Like other initiator caspases, its activation is triggered by the assembly of a
multimeric protein complex. This complex is known as the inflammasome, which
resembles structurally the apoptosome. In this complex, NLR (NACHT-LRR) family
members such as NALP1, 3, and IPAF serve as scaffolding proteins (Martinon and
Tschopp, 2005). The activation of caspase--l in the inflammasome is comparable to that
of caspase-9 in the apoptosome (Figure 3). In general, NLR members are typically
composed of three domains: a Leucine Rich Repeat (LRR) ligand sensing domain, a
NACHT nucleotide binding and oligomerization domain, and a CARD or Pyrin domain
(PYD) for caspase recruitment (Martinon and Tschopp, 2004; Nadiri et al., 2006).
Four types of inflammasomes have been characterized and shown to activate caspase-l:
NALP-l, NALP-3, NAIP5 and IPAF. (Martinon and Tschopp, 2004; Molofsky et al.,
2006). The interaction between NALP proteins and caspase-l is not direct but depends on
the adaptor ASC; whereas, IPAF can activate caspase-l directly via its CARD
11
(Mariathasan et al., 2006). Recently some natural stimuli of the inflammasome have been
identified. The NALP-3 inflammasome stimuli include bacterial RNA ((Kanneganti et a l ,
2006), uric acid crystals associated with Gout (Martinon et al., 2006), extracellular ATP,
the toxin Nigericin and the calcium channel affecting marine toxin maitotoxin (Zamboni
et al., 2006); the NAIP5 inflammasome is stimulated by the bacteria Legionella
pneumophila (Fortier et al., 2006; Zamboni et al., 2006); The IPAF inflammasome is
activated by Salmonella (Mariathasan et al., 2004) and by cytoplasmic flagellin (Miao et
al , 2006).
More recently, elegant work
by Alnemri and colleagues
have introduced a second
caspase-1 activating complex,
which they termed the
"pyroptosome". The
pyroptosome is solely
composed of ASC dimers and
is devoid of any NLR. It is
hypothesized when the
C«fl «tr&«£iN&imd$a
Apoptosomw M Apaf-1
Cja<spas*»S
lUUNOEFCMWNAUM
i n
CMPMMMI n-pto4L*1|k prefflL-19
processing and smctetion
IjInfinmmMtian • Apoploslsj
Figure 3 Apoptosome vs. Inflammasome (Creagh, Conroy et al. 2003)
activation of caspase-1 in the inflammasome is subtle, it results in controlled
inflammation; while activation of caspase-1 by pyroptosome is more complete, which
leads to cell death or pyroptosis. We hypothesize that pyroptosis is executed by the
"hyperactive" caspase-1 through cleaving a wider range of cellular substrates.
Caspase-1 function
Caspase-1 was originally known as the interleukin-ip-converting enzyme (ICE), as it
processes pro-IL-l|3 to its active form. Now we know that it also acts on IL-18 and IL-33.
12
(Mariathasan et al., 2006). Recently sorne natural stimuli of the inflammasome have been
identified. The NALP-3 inflammasome stimuli include bacterial RNA «Kanneganti et al.,
2006), uric acid crystals associated with Gout (Martinon et al., 2006), extracellular ATP,
the toxin Nigericin and the calcium channel affecting marine toxin maitotoxin (Zamboni
et al., 2006); the NAIP5 inflammasome is stimulated by the bacteria Legionella
pneumophila (Fortier et al., 2006; Zamboni et al., 2006); The IPAF inflammasome is
activated by Salmonella (Mariathasan et al., 2004) and by cytoplasmic flagellin (Miao et
al.,2006).
More recently, elegant work
by Alnemri and colleagues
have introduced a second
caspase-l activating complex,
which they termed the
"pyroptosome". The
pyroptosome IS solely
composed of ASC dimers and
is devoid of any NLR. It is
hypothesized when the
.It" '" Cytœh_.,
1 Bec!leriallnftlctiGn 1
la ~4 •
•••• "'. • 4lc·IllI···~-15"1ID1_'" t41111ml1Blœ1"'.-
~ ..,...11.-1" p ...... L-18 ~ingr~
[~tOeiBl E~i!;i)
Figure 3 Apoptosome vs. Inflammasome (Creagh, Conroy et al. 2003)
activation of caspase-l in the inflammasome is subtle, it results III controlled
inflammation; while activation of caspase-l by pyroptosome is more complete, which
leads to cell death or pyroptosis. We hypothesize that pyroptosis is executed by the
"hyperactive" caspase-l through cleaving a wider range of cellular substrates.
Caspase-l junetion
Caspase-l was originally known as the interleukin-l p-converting enzyme (ICE), as it
processes pro-IL-lp to its active form. Now we know that it also acts on IL-18 and IL-33.
12
The importance of caspase-1 in inflammation is highlighted by the fact that the cytokines
that it activates have been implicated in the pathophysiology of various diseases,
including rheumatoid arthritis, septic shock and inflammatory bowel disease (Dinarello
and Wolff, 1993). The role of caspase-1 in inflammation is fully supported by the in vivo
study that caspase-1 knockout mice are defective in the production of mature IL-1(3,
IL-la and IL-18 as well as the downstream cytokines IL-6 and interferon-y, and are
highly resistant to endotoxic shock (Kuida et al., 1995; Li et al., 1995). On the contrary,
these mice as expected are susceptible to bacterial infections with E. coli and Salmonella
(Joshi et al., 2002; Lara-Tejero et al., 2006).
Caspase-1 is not involved in apoptosis due to the fact that caspase-1-deficient mice have
no apparent defects in developmental programmed cell death and caspase-1 deficient
cells respond normally to most apoptotic stimuli (Earnshaw et al., 1999). However,
caspase-1 activation in macrophages infected with Salmonella or Shigella causes massive
cell death in host (Fink and Cookson, 2006). This form of cell death is termed as
pyroptosis as it is related to inflammation and is distinct from apoptosis, which actively
inhibits inflammation.
Except for caspase-1 dependent cell death in the immune system, caspase-1 function is
also implicated in cell death in central neuron system and cardiac circulation systems.
Mice deficent in caspase-1 are resistant to neonatal hypoxic-ischemic brain damage (Liu
et al., 1999). Transgenic mice that express a catalytically inactive caspase-1 (C285G)
mutant inhibit trophic factor withdrawal-induced apoptosis in dorsal root cells; similar
results were obtained in neurons isolated from newborn caspase-1 knockout mice
(Friedlander et al., 1997b). As well, transgenic mice with overexpresion of caspase-1 in
heart have shown strikingly increased cardiac myocyte cell death under
ischemia/reperfusion injury (Syed et al., 2005). This study demonstrates that
13
The importance of caspase-l in inflammation is highlighted by the fact that the cytokines
that it activates have been implicated in the pathophysiology of various diseases,
inc1uding rheumatoid arthritis, septic shock and inflammatory bowel disease (Dinarello
and Wolff, 1993). The role of caspase-l in inflammation is fully supported by the in vivo
study that caspase-l knockout mice are defective in the production of mature IL-l (3,
IL-l a and IL-18 as weIl as the downstream cytokines IL-6 and interferon-y, and are
highly resistant to endotoxic shock (Kuida et al., 1995; Li et al., 1995). On the contrary,
these mi ce as expected are susceptible to bacterial infections with E. coli and Salmonella
(Joshi et al., 2002; Lara-Tejero et al., 2006).
Caspase-l is not involved in apoptosis due to the fact that caspase-l-deficient mice have
no apparent defects in developmental programmed cell death and caspase-l deficient
cells respond normally to most apoptotic stimuli (Earnshaw et al., 1999). However,
caspase-l activation in macrophages infected with Salmonella or Shigella causes massive
cell death in host (Fink and Cookson, 2006). This form of cell death is termed as
pyroptosis as it is related to inflammation and is distinct from apoptosis, which actively
inhibits inflammation.
Except for caspase-l dependent cell death in the immune system, caspase-l function is
also implicated in cell death in central neuron system and cardiac circulation systems.
Mice deficent in caspase-l are resistant to neonatal hypoxic-ischemic brain damage (Liu
et al., 1999). Transgenic mice that express a catalytically inactive caspase-l (C285G)
mutant inhibit trophic factor withdrawal-induced apoptosis in dorsal root ceIls; similar
results were obtained in neurons isolated from newborn caspase-l knockout mice
(Friedlander et al., 1997b). As weIl, transgenic mice with overexpresion of caspase-l in
heart have shown strikingly increased cardiac myocyte cell death under
ischemia/reperfusion injury (Syed et al., 2005). This study demonstrates that
13
cardiomyocytes can respond to caspase-1 overexpression and its subsequent activation to
initiate cell death under certain stresses.
In contrast to pyroptosis caused by activation of caspase-1 in macrophages, a novel
function of caspase-1 has been proposed in cell survival (Saleh, 2006). In response to a
drop in cytosolic [k+], caspase-1 is activated via IPAF and ASC/NALP3 inflammasomes,
followed by activation of central regulators of membrane biogenesis, the Sterol
Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival
(Gurcel et al., 2006). Interestingly, other caspases are also implicated in this pathway:
SREBP1/2 were shown to be cleaved and activated by caspases (Wang et al., 1995; Wang
et al., 1996) and Caspase-2 was found to be a transcriptional target of SREBP2 (Logette
et al., 2005). These findings link lipid metabolism to cell death and reveal a mechanism
by which cells regulate the balance between survival and death pathways in response to
pathogen invasion.
Negative regulation of caspase-1 by caspase-12
Caspase-12 has been shown to negatively regulate caspase-1 both in humans and rodents
(Saleh et al., 2004). A polymorphism in caspase-12 results in the production of either a
truncated variant (caspase-12 S) or a full-length variant (caspase-12L). Most individuals
express caspase-12S, and only about 20% of individuals of African descent express
caspase-12L. Those individuals expressing caspase-12L have hypo-responsiveness to
LPS-mediated production of cytokines such as IL-ip and INFy and have an increased risk
of developing severe sepsis (Saleh et al., 2004). Like the human full-length variant of
caspase-12, murine caspase-12 also abrogates the inflammatory response (Saleh et al.,
2006). Caspase-12 deficient mice are more resistant to sepsis and are able to clear
bacterial pathogens more efficiently than wild-type mice. This may largely be due to the
inhibitory effect caspase-12 has on caspase-1 (Scott and Saleh, 2007).
14
cardiomyocytes can respond to caspase-l overexpression and its subsequent activation to
initiate cell death under certain stresses.
In contrast to pyroptosis caused by activation of caspase-l in macrophages, a novel
function of caspase-l has been proposed in cell survival (Saleh, 2006). In response to a
drop in cytosolic [k+], caspase-l is activated via IPAF and ASC/NALP3 inflammasomes,
followed by activation of central regulators of membrane biogenesis, the Sterol
Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival
(Gurcel et al., 2006). Interestingly, other caspases are also implicated in this pathway:
SREBPI/2 were shown to be cleaved and activated by caspases (Wang et al., 1995; Wang
et al., 1996) and Caspase-2 was found to be a transcriptional target of SREBP2 (Logette
et al., 2005). These findings link lipid metabolism to cell death and reveal a mechanism
by which cells regulate the balance between survival and death pathways in response to
pathogen invasion.
Negative regulation of caspase-l hy caspase-12
Caspase-12 has been shown to negatively regulate caspase-l both in humans and rodents
(Saleh et al., 2004). A polymorphism in caspase-12 results in the production of either a
truncated variant (caspase-12S) or a full-Iength variant (caspase-12L). Most individuals
express caspase-12S, and only about 20% of individuals of African descent express
caspase-12L. Those individuals expressing caspase-12L have hypo-responsiveness to
LPS-mediated production of cytokines such as IL-l p and INFy and have an increased risk
of developing severe sepsis (Saleh et al., 2004). Like the human full-Iength variant of
caspase-12, murine caspase-12 also abrogates the inflammatory response (Saleh et al.,
2006). Caspase-12 deficient mice are more resistant to sepsis and are able to clear
bacterial pathogens more efficiently than wild-type mice. This may largely be due to the
inhibitory effect caspase-12 has on caspase-l (Scott and Saleh, 2007).
14
Caspase-1 substrates
Caspase-1 is an inflammatory caspase that is responsible for maturation of
pro-inflammatory cytokines. Although, in vitro, caspase-1 was shown to process the
kinase PITSLRE (Beyaert et al., 1997), actin (Chen et al , 1996), parkin (Kahns et al,
2003) and PARP (Gu et al., 1995), cleavage of these substrates occurs by other caspases
in vivo. Pro-IL-l(3 and pro-IL-18 are the best-known caspase-1 substrates. They are
synthesized as biologically inactive forms, which lack a leader peptide. It has been
suggested that the precursors of IL-113 and IL-18 enter the specialized secretory lysosome
together with components of the inflammasome, following caspase-1 activation. The
secretory lysosome fuses with the cell membrane and the caspase-1-processed cytokines
are released as active cytokines (Pizzirani et al., 2006). IL-33 has recently been identified
as a caspase-1 substrate. Mature IL-33 binds to ST2 (an orphan IL-1 receptor) and results
in the activation of NF-KB and MAPK that drive production of type 2 cytokines (e.g.
IL-4, IL-5 and IL-13) from polarized Th2 cells. The induction of these type 2 cytokines
by IL-33 in vivo is attributed to severe pathological changes observed in mucosal organs
(Schmitz et al , 2005).
We now known that caspase-1 has roles in cell death as well as in cell survival in
addition to its well-established role in the maturation of pro-inflammatory cytokines. To
fulfill these additional roles, caspase-1 must cleave proteins other than the precursors of
IL-1 (3, IL-18 and IL-33. The identification of caspase-1 substrates is essential for our
understanding of its wide range of actions.
15
Caspase-l s ubstrates
Caspase-l is an inflammatory caspase that is responsible for maturation of
pro-inflammatory cytokines. Although, in vitro, caspase-1 was shown to process the
kinase PITSLRE (Beyaert et al., 1997), actin (Chen et al., 1996), parkin (Kahns et al.,
2003) and PARP (Gu et al., 1995), cleavage of these substrates occurs by other caspases
in vivo. Pro-IL-1f3 and pro-IL-18 are the best-known caspase-1 substrates. They are
synthesized as biologically inactive forms, which lack a leader peptide. It has been
suggested that the precursors of IL-l 13 and IL-18 enter the specialized secretory lysosome
together with components of the inflammasome, following caspase-1 activation. The
secretory lysosome fuses with the cell membrane and the caspase-1-processed cytokines
are released as active cytokines (Pizzirani et al., 2006). IL-33 has recently been identified
as a caspase-1 substrate. Mature IL-33 binds to ST2 (an orphan IL-1 receptor) and results
in the activation of NF-KB and MAPK that drive production of type 2 cytokines (e.g.
IL-4, IL-5 and IL-13) from polarized Th2 cells. The induction of these type 2 cytokines
by IL-33 in vivo is attributed to severe pathological changes observed in mucosal organs
(Schmitz et al., 2005).
We now known that caspase-1 has roles in cell death as well as in cell survival in
addition to its well-established role in the maturation of pro-inflammatory cytokines. To
fulfill the se additional roles, caspase-1 must cleave proteins other than the precursors of
IL-1f3, IL-18 and IL-33. The identification of caspase-l substrates is essential for our
understanding of its wide range of actions.
15
GOAL OF THIS STUDY
Cleavage of caspase substrates is central to the function of caspases. To date, more than
280 caspase substrates have been identified. (Fischer et al., 2003). The consequence of
substrate cleavage greatly advanced our understanding of the role of caspases in
apoptosis. As we know, caspase-3 has a predominant role in apoptosis, but growing
evidence indicates a non-apoptotic role of caspase-3 as well (Launay et al., 2005). The
identification of novel caspase substrates would provide us with insights into caspase
functions both in apoptosis and non-apoptotic processes.
Unlike caspase-3, only few caspase-1 substrates have been identified, despite the fact that
it was the first caspase to be cloned. These substrates include the pro-inflammatory
cytokines pro-IL-ip and pro-IL-18, giving caspase-1 its designation as an "inflammatory
caspase". Caspase-1, however, has been shown to induce cell death, namely macrophage
pyroptosis in response to bacterial infection (Monack et al., 2001). Recently, the
mechanism by which caspase-1 is activated has been characterized but the precise
mechanism(s) by which caspase-1 initiates/executes cell death is still unclear.
Identification of caspase-1 substrates is thus critical for completing our knowledge in this
respect.
The diagonal gel approach is a proteomic technique for screening for protease substrates.
In this study, we applied this approach to identify substrates of caspases-1 and -3.
16
GOAL OF THIS STUDY
Cleavage of caspase substrates is central to the function of caspases. To date, more than
280 caspase substrates have been identified. (Fischer et al., 2003). The consequence of
substrate cleavage greatly advanced our understanding of the role of caspases in
apoptosis. As we know, caspase-3 has a predominant role in apoptosis, but growing
evidence indicates a non-apoptotic role of caspase-3 as well (Launay et al., 2005). The
identification of novel caspase substrates would provide us with insights into caspase
functions both in apoptosis and non-apoptotic processes.
Unlike caspase-3, only few caspase-I substrates have been identified, despite the fact that
it was the first caspase to be cloned. These substrates include the pro-inflammatory
cytokines pro-IL-I~ and pro-IL-18, giving caspase-I its designation as an "inflammatory
caspase". Caspase-I, however, has been shown to induce cell death, namely macrophage
pyroptosis in response to bacterial infection (Monack et al., 200 I). Recently, the
mechanism by which caspase-I is activated has been characterized but the precise
mechanism(s) by which caspase-I initiates/executes cell death is still unclear.
Identification of caspase-I substrates is thus critical for completing our knowledge in this
respect.
The diagonal gel approach is a proteomic technique for screening for protease substrates.
In this study, we applied this approach to identify substrates of caspases-I and -3.
16
MATERIALS AND METHODS
Cell culture
THP-1 cells were cultured in RPMI 1640 (Invitrogen) supplemented with L-glutamine,
10 % heat inactivated fetal bovine serum (FBS) and penicillin (100 U/ml) and
streptomycin (100[Ag/ml). Cells were grown in a humidified incubator at 37° C, with 5%
CO2 and maintained at density 1 X 106 to 1.5 X 106. Confluent Cells were split by
diluting 1: 3 with fresh culture media.
HEK293T and HeLa cells were cultured in DMEM (Invitrogen) supplemented with
L-glutamine, 10 % heat inactivated fetal bovine serum supplemented with penicillin (100
U/ml) and streptomycin (100ug/ml). Cells were grown in a humidified incubator at 37° C
with 5% CO2. Confluent cells were split by trypsinization and by seeding 2.5 x 106 to a
new 10 cm cell culture dish
Human PBMC isolation and proliferation
Human PBMCs were isolated from human blood cells by removing red blood cells using
RBC lysis buffer (sigma). 2 x 107 cells were cultured in 15 ml RPMI. For proliferation,
the cells were treated with 1 u.g/ml anti-CD3 antibody and 20 U/ml human recombinant
IL-2 and cultured for 4 days in a humidified incubator at 37° C with 5% CO2.
Diagonal gel
THP-1 cells or human PBMCs were lysed in SDS Laemmli loading buffer (50 mM Tris
[pH 6.8], 2% SDS, 0.1% bromophenol blue, 10% glycerol, 2.5% (3-mercaptoethanol) and
sonicated. 400 ug of protein was resolved by SDS-PAGE 10% (first dimension). After
migration, the lane containing the protein was excised and soaked in 40% EtOH and 10%)
17
MATERIALS AND METHODS
Cell culture
THP-1 cens were cultured in RPMI 1640 (lnvitrogen) supplemented with L-glutamine,
10 % heat inactivated fetai bovine serum (FBS) and penicillin (100 U/mI) and
streptomycin (IOO!-tg/ml). cens were grown in a humidified incubator at 37° C, with 5%
C02 and maintained at density 1 X 106 to 1.5 X 106. Confluent cens were split by
diluting 1: 3 with fresh culture media.
HEK293T and HeLa cells were cultured in DMEM (lnvitrogen) supplemented with
L-glutamine, 10 % heat inactivated fetaI bovine serum supplemented with penicillin (100
U/ml) and streptomycin (100!-tg/ml). cens were grown in a humidified incubator at 37° C
with 5% CO2. Confluent cens were split by trypsinization and by seeding 2.5 x 106 to a
new 10 cm cen culture dish
Human PBMC isolation and proliferation
Human PBMCs were isolated from human blood cens by removing red blood cens using
RBC lysis buffer (sigma). 2 x 107 cens were cultured in 15 ml RPMI. For proliferation,
the cens were treated with l!-tg/ml anti-CD3 antibody and 20 U/ml human recombinant
IL-2 and cultured for 4 days in a humidified incubator at 37° C with 5% CO2.
Diagonal gel
THP-I cens or human PBMCs were lysed in SDS Laemmli loading buffer (50 mM Tris
[pH 6.8], 2% SDS, 0.1 % bromophenol blue, 10% glycerol, 2.5% f)-mercaptoethanol) and
sonicated. 400 !-tg of protein was resolved by SDS-PAGE 10% (first dimension). After
migration, the lane containing the prote in was excised and soaked in 40% EtOH and 10%
17
Acetic acid for 10 min, in EtOH 30% for 10 min, and then in ultrapure water for 2 x 10
min. The lane was then air dried until the gel started to curl and then soaked in CHEGG
buffer (50 mM Hepes-KOH [PH 7.2], 10% glycerol, 0.1% chaps, 2 mM EDTA and 5
mM DTT freshly added) with or without 50 jig of recombinant caspase-1 or caspase-3
(Merck), and incubated overnight at 37°C. The lane was washed in water to remove the
excess protease and then incubated in SDS Laemmli loading buffer for 10 min at 95°C.
After cooling, the lane was then loaded on a second 10% SDS PAGE gel and resolved
again. After migration, the gel was stained using Sypro Ruby (Invitrogen) or silver stain.
Cleaved proteins, which were located under the diagonal, were excised from the gel and
identified by mass spectrometry (at the McGill University and Genome Quebec
Innovation Centre using LC-MS).
In vitro cleavage assay with whole cell lysates
Each 8 x 106 THP-1 cells were lysed in 100 ul CHEGG buffer and sonicated. Protein
concentration was measured using Bio-Rad protein assay reagent. Each 40 ug proteins
were incubated with 340 ng recombinant caspase-1 (Merck) or caspase-3 (Sigma) at 37
°C for 3 hrs, then resolved on 4-12% SDS-PAGE for western blots.
Western blot
After SDS-PAGE, the proteins on the gels were transferred to Hybond-C Extra,
nitrocellulose membrane (Amersham) at 50 V for 1 hr. The blots were first incubated in
blocking buffer (Tris-buffered saline, pH 7.4 (TBS), 5% (w/v) non fat milk, 0.1 % (v/v)
Tween 20) for 1 hr at room temperature and then incubated for 2 hrs in primary antibody
diluted in the same buffer. After washing three times each for 10 min in 1 xTBS with
0.1% (v/v) Tween 20 for 10 min, blots were incubated for 1 h at room temperature with
secondary antibodies that were diluted in blocking buffer. Blots were washed three times
in 1 x TBS, 0.2 % (v/v) Tween 20 for 5 min, and three times in 1 x TBS, 0.1% (v/v)
18
Acetic acid for 10 min, in EtOH 30% for 10 min, and then in ultrapure water for 2 x 10
min. The lane was then air dried until the gel started to curl and then soaked in CHEGG
buffer (50 mM Hepes-KOH [PH 7.2], 10% glycerol, 0.1 % chaps, 2 mM EDTA and 5
mM DTT freshly added) with or without 50 !-tg of recombinant caspase-1 or caspase-3
(Merck), and incubated ovemight at 37°C. The lane was washed in water to remove the
excess protease and then incubated in SDS Laemmli loading buffer for 10 min at 95°C.
After cooling, the lane was then loaded on a second 10% SDS PAGE gel and resolved
again. After migration, the gel was stained using Sypro Ruby (Invitrogen) or silver stain.
Cleaved proteins, which were located under the diagonal, were excised from the gel and
identified by mass spectrometry (at the McGill University and Genome Quebec
Innovation Centre using LC-MS).
In vitro cleavage assay with whole cel/lysates
Each 8 x 106 THP-1 cells were lysed in 100 !-t1 CHEGG buffer and sonicated. Protein
concentration was measured using Bio-Rad protein assay reagent. Each 40 !-tg proteins
were incubated with 340 ng recombinant caspase-1 (Merck) or caspase-3 (Sigma) at 37
oC for 3 hrs, then resolved on 4-12% SDS-PAGE for western blots.
Western blot
After SDS-PAGE, the proteins on the gels were transferred to Hybond-C Extra,
nitrocellulose membrane (Amersham) at 50 V for 1 hr. The blots were first incubated in
blocking buffer (Tris-buffered saline, pH 7.4 (TBS), 5% (w/v) non fat milk, 0.1 % (v/v)
Tween 20) for 1 hr at room temperature and then incubated for 2 hrs in primary antibody
diluted in the Same buffer. After washing three times each for 10 min in 1 xTBS with
0.1 % (v/v) Tween 20 for 10 min, blots were incubated for 1 h at room temperature with
secondary antibodies that were diluted in blocking buffer. Blots were washed three times
in 1 x TBS, 0.2 % (v/v) Tween 20 for 5 min, and three times in 1 x TBS, 0.1 % (v/v)
18
Tween 20 for 5 min. Detection was performed by chemiluminescence using ECL
(Amersham).
The following antibodies were used: anti- cc-enolase, aldolase, triosephosphate isomerase
and caspase-1 P10 (Santa Cruz); anti- GAPDH (Abeam); anti- a-tubulin (sigma), anti-
IL-ip (cell signaling).
RNA extraction andRT-PCR
Total RNA was isolated from THP-1 cells using the Trizol Reagent (Invitrogen). Each 1
x 106 cells were lysed in 200 ul Trizol, and extracted with 40 ul chloroform. Following
centrifugation, RNA remained in the upper aqueous phase. The upper layer was removed
to a fresh tube and isopropanol was added to precipitate the RNA. The RNA was
dissolved in 50 ul DEPC water, and its concentration was measured by spectrometry. For
first strand cDNA synthesis, 200 ng of total RNA was incubated with 1 ?g oligo (dT)i2-is
primer (500 ?g/ml, Invitrogen), 1 ul lOmM dNTP mix in a final volume of 12 ul. The
reaction was heated to 65 °C for 5 min and then quickly chilled on ice to allow annealing
of the oligo (dT). The mixture was further incubated at 42 °C for 2 min in IX
First-Strand Buffer (supplied with the kit), 0.01 M DTT and 40 U Rnase inhibitor
(Promega). M-MLV RT (Invitrogen) was added to the mixture and incubated for 50 min
at 37 °C, followed by enzyme inactivation at 70°C for 15 min.
Constructs and site-directed mutagenesis
PCR amplification of glycolysis enzymes from cDNA was carried out by mixing Taq
DNA polymerase (Roche) 2.5 units, 1.5 mM MgCl2 0.8 mM dNTP, 5 ul template cDNA
and 0.8 uM forward and reverse primers specific for glycolysis enzymes (table 2) in a
final volume of 50 ul. PCR cycling was 2 min at 94 °C for initial denaturing, followed by
10 cycles of 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 60 s and 20 cycles of 94 °C for
19
Tween 20 for 5 min. Detection was performed by chemiluminescence using ECL
(Amersham).
The following antibodies were used: anti- a-enolase, aldolase, triosephosphate isomerase
and caspase-1 PlO (Santa Cruz); anti- GAPDH (Abcam); anti- a-tubulin (sigma), anti
IL-1 f3 (cell signaling).
RNA extraction and R T-PCR
Total RNA was isolated from THP-1 cells using the Trizol Reagent (Invitrogen). Each 1
x 106 cells were lysed in 200 !lI Trizol, and extracted with 40 !lI chloroform. Following
centrifugation, RNA remained in the upper aqueous phase. The upper layer was removed
to a fresh tube and isopropanol was added to precipitate the RNA. The RNA was
dissolved in 50 !lI DEPC water, and its concentration was measured by spectrometry. For
first strand cDNA synthesis, 200 ng of total RNA was incubated with 1 ?g oligo (dT)12-18
primer (500 ?g/ml, lnvitrogen), 1 !lI lOmM dNTP mix in a final volume of 12 !lI. The
reaction was heated to 65 oC for 5 min and then quickly chilled on ice to allow annealing
of the oligo (dT). The mixture was further incubated at 42 oC for 2 min in IX
First-Strand Buffer (supplied with the kit), 0.01 M DTT and 40 U Rnase inhibitor
(Promega). M-MLV RT (Invitrogen) was added to the mixture and incubated for 50 min
at 37 oC, followed by enzyme inactivation at 70°C for 15 min.
Constructs and site-directed mutagenesis
PCR amplification of glycolysis enzymes from cDNA was carried out by mixing Taq
DNA polymerase (Roche) 2.5 units, 1.5 mM MgCh 0.8 mM dNTP, 5 !lI template cDNA
and 0.8 !lM forward and reverse primers specifie for glycolysis enzymes (table 2) in a
final volume of 50 !lI. PCR cycling was 2 min at 94 oC for initial denaturing, followed by
10 cycles of 94 oC for 30 s, 60 oC for 60 s, and 72 OC for 60 s and 20 cycles of 94 oC for
19
30 s, 60 °C for 60 s, and 72 °C for 60 s with a elongation of 20 s for each cycle and then
elongation at 72 °C for 7 min. The PCRs were cloned to pcDNA3.1+ (neo) vector. The
constructs from each clone were confirmed by sequencing.
Potential cleavage site mutations were introduced by site-directed mutagenesis using the
QuickChange site-directed mutagenesis kit (Stratagene) following the manufacturer's
instructions. The primers for mutagenesis were summarized in table 3. All mutations
were confirmed by sequencing.
In vitro cleavage of S methionine-labeled substrates i f
S Methionine-labeled substrates were obtained by coupled in vitro transcription and
translation using the Promega TNT reticulocyte lysate system. 0.7 ug of the cDNA
constructs were incubated with T7 polymerase, rabbit reticulocyte lysates, amino acid
mixture minus methionine, and 35S methionine for 1.5 h at 30 °C. Cleavage of the in vitro
transcribed and translated 35S labeled products were performed by incubation at 37 °C for
4 hrs in the presence or absence of purified human recombinant caspase-1 (Merck) in
CHEGG buffer. The cleavage reaction was terminated by the addition of SDS Laemmli
loading buffer and resolved by SDS-PAGE, and viewed by autoradiography.
Caspase-1 activation with ATP/nigericin treatment
THP-1 cells were pre-treated with 20 ng PMA for 24 hrs, and then were treated with 100
ug LPS for overnight, followed by 20 min incubation with 5mM ATP or 20 ^M nigericin
and then the culture media were replaced with fresh media. The cells were cultured for an
additional 3 hrs for IL-1(3 secretion. Cells were lysed in Laemmli loading buffer and
resolved in 4-12 % SDS PAGE for Western blot.
20
30 s, 60 oC for 60 s, and 72 oC for 60 s with a elongation of 20 s for each cycle and then
elongation at 72 oC for 7 min. The PCRs were cloned to pcDNA3.1+ (neo) vector. The
constructs from each clone were confirmed by sequencing.
Potential cleavage site mutations were introduced by site-directed mutagenesis using the
QuickChange site-directed mutagenesis kit (Stratagene) folIowing the manufacturer's
instructions. The primers for mutagenesis were summarized in table 3. AlI mutations
were confirmed by sequencing.
In vitro cleavage Of35S methionine-labeled substrates
35S Methionine-Iabeled substrates were obtained by coupled in vitro transcription and
translation using the Promega TNT reticulocyte lysate system. 0.7 /lg of the cDN A
constructs were incubated with T7 polymerase, rabbit reticulocyte lysates, amino acid
mixture minus methionine, and 35S methionine for 1.5 h at 30 oC. Cleavage of the in vitro
transcribed and translated 35S labeled products were performed by incubation at 37 oC for
4 hrs in the presence or absence of purified human recombinant caspase-l (Merck) in
CHEGG buffer. The cleavage reaction was terminated by the addition of SDS Laemmli
loading buffer and resolved by SDS-PAGE, and viewed by autoradiography.
Caspase-l activation with ATP/nigericin treatment
THP-l celIs were pre-treated with 20 ng PMA for 24 hrs, and then were treated with 100
ug LPS for overnight, followed by 20 min incubation with 5mM ATP or 20 /lM nigericin
and then the culture media were replaced with fresh media. The celIs were cultured for an
additional 3 hrs for IL-l Il secretion. CelIs were lysed in Laemmli loading buffer and
resolved in 4-12 %SDS PAGE for Western blot.
20
THP-1 cells Salmonella infection
Bacterial preparation for infection
Wild-type bacteria Salmonella (SL1344) were cultured aerobically at 37°C in
Luria-Bertani (LB) broth or on LB agar without antibiotics. Bacteria were freshly plated
on LB agar. To obtain stationary-phase bacteria for infection of THP-1 cells, LB broth
was inoculated with a single colony and grown overnight with vigorous shaking. Before
infection, bacteria were diluted 1:10 in LB broth, grew until OD600 0.9 (equivalent to 105
bacteria/ml), and then bacteria were harvested by centrifugation, washed with PBS, and
incubated for 20 min at 37°C in THP-1 culture media (without antibiotics). The bacteria
were used immediately for infection of THP-1 cells.
Cell culture and infection.
THP-1 cells were maintained at a density of 1 x 105 to 2 x 106 cells/ml. One day prior to
infection, the cells were harvested, washed, and resuspended in fresh medium without
antibiotics. The cells were incubated with PMA (20 ng/ml) for one overnight to
differentiate the cells into adherent macrophage-like cells, The next day, medium and
nonadherent cells were removed and replaced with fresh complete medium without
antibiotics. The cells were then primed with 50 ng/ml crude LPS for one overnight. The
next day, the cells were infected with prepared bacteria Salmonella at a multiplicity of
infection (moi) of 10:1 bacteria: THP-1 cells. Culture plates were centrifuged at 500 x g
for 10 min and incubated at 37°C for 30 min to allow phagocytosis to occur. Under these
conditions, essentially all cells are infected with bacteria. The medium was then replaced
with fresh medium without antibiotics and incubated for additional 16 hrs. Two parallel
wells were prepared for FLICAcasp_1 analysis and western blot for each condition. The
cells for western blot were lysed in SDS Laemmli loading buffer and resolved on 4-12 %
SDS PAGE.
21
THP-l cells Salmonella infection
Bacterial preparation for infection
Wild-type bacteria Salmonella (SL1344) were cultured aerobically at 37°C in
Luria-Bertani (LB) broth or on LB agar without antibiotics. Bacteria were freshly plated
on LB agar. To obtain stationary-phase bacteria for infection of THP-1 cells, LB broth
was inoculated with a single colony and grown overnight with vigorous shaking. Before
infection, bacteria were diluted 1: 1 0 in LB broth, grew until OD600 0.9 (equivalent to 105
bacteria/ml), and then bacteria were harvested by centrifugation, washed with PBS, and
incubated for 20 min at 37°C in THP-1 culture media (without antibiotics). The bacteria
were used immediately for infection of THP-I cells.
Cell culture and infection.
THP-1 cells were maintained at a density of 1 x 105 to 2 x 106 cells/ml. One day prior to
infection, the cells were harvested, washed, and resuspended in fresh medium without
antibiotics. The cells were incubated with PMA (20 ng/ml) for one overnight to
differentiate the cells into adherent macrophage-like cells, The next day, medium and
nonadherent cells were removed and replaced with fresh complete medium without
antibiotics. The cells were then primed with 50 ng/ml crude LPS for one overnight. The
next day, the cells were infected with prepared bacteria Salmonella at a multiplicity of
infection (moi) of 10: 1 bacteria: THP-1 cells. Culture plates were centrifuged at 500 x g
for 10 min and incubated at 37°C for 30 min to allow phagocytosis to occur. Under the se
conditions, essentially aIl cells are infected with bacteria. The medium was then replaced
with fresh medium without antibiotics and incubated for additional 16 hrs. Two parallel
wells were prepared for FLICAcasp-l analysis and western blot for each condition. The
cells for western blot were lysed in SDS Laemmli loading buffer and resolved on 4-12 %
SDS PAGE.
21
FLICAcasp~ activity measurement
FLICAcasp"1 (immunochemistry Technologies) was added to cell culture at the indicated
time points, followed by 1 hr incubation at 37 °C. Cells were collected and washed two
times with wash buffer (supplied with the kit) and then re-suspended in the same buffer.
Fluorescence was measured by FACS (BD FACS Calibur) under the FL1 channel.
Results were analyzed using the software FlowJo.
LDH measurement ofTHP-1 cells infected with Salmonella
THP-1 cells were aliquoted into 10 cm plates at a density of 2 X 106 cells per plate. The
cells were treated with PMA (20 ng/ml) for one overnight. After PMA treatment, the
media were replaced with fresh media. The cells were infected with Salmonella (moi
1:100) following the Salmonella infection procedure. After 4 hrs of infection, the culture
media were collected for LDH measurement using a kit from Trinity Biotech following
its instruction. For YVAD-FMK treatment, the cells were pre-treated with YVAD-FMK
(100 \\M) 30 min before PMA treatment, and YVAD-FMK was kept in the culture media
at all time.
Mice model of LPS-induced septic shock
C57BL/6 mice (8-12 weeks old) were injected intraperitoneally with 20mg/kg LPS from
Escherichia coli (sterotype 055:B5, Sigma). Control mice were injected intraperitoneally
with saline. The mice were killed 12 or 24 hrs post-injection. Diaphragm muscle was
excised and proteins were extracted for western blot.
22
FLICAcasp-l activity measurement
FLICAcasp-1 (immunochemistry Technologies) was added to cell culture at the indicated
time points, followed by 1 br incubation at 37°C. Cells were collected and washed two
times with wash buffer (supplied with the kit) and then re-suspended in the same buffer.
Fluorescence was measured by FACS (BD FACS Calibur) under the FLI channel.
Results were analyzed using the software FlowJo.
LDH measurement of THP-l eells infeeted with Salmonella
THP-l cells were aliquoted into 10 cm plates at a density of 2 X 106 cells per plate. The
cells were treated with PMA (20 ng/ml) for one overnight. After PMA treatment, the
media were replaced with fresh media. The cells were infected with Salmonella (moi
1: 100) following the Salmonella infection procedure. After 4 hrs of infection, the culture
media were collected for LDH measurement using a kit from Trinity Biotech following
its instruction. For YVAD-FMK treatment, the cells were pre-treated with YVAD-FMK
(100 ~M) 30 min before PMA treatment, and YVAD-FMK was kept in the culture media
at aIl time.
Mice model of LPS-indueed septie shock
C57BL/6 mice (8-12 weeks old) were injected intraperitoneally with 20mg/kg LPS from
Escherichia coli (sterotype 055:B5, Sigma). Control mice were injected intraperitoneally
with saline. The mice were killed 12 or 24 hrs post-injection. Diaphragm muscle was
excised and proteins were extracted for western blot.
22
RESULTS AND DISCUSION
/. Caspases-1 and-3 substrates identified by the diagonal gel approach
The diagonal gel approach is a technique for protease substrate screening. It was recently
applied to identify the mitochondrial caspase-3 substrate NDUFS (Ricci et al., 2004).
Figure 4 schematically outlines this method. In general, whole cell lysates are separated
on a 1st dimension SDS PAGE gel. The gel lane is cut, dehydrated and then rehydrated in
a buffer containing the protease of interest. These steps wash out the SDS and allow the
protease to diffuse through the gel pores and digest its specific substrates. Following this
in-gel digestion, the gel lane is loaded horizontally on a second SDS-PAGE gel and the
proteins are resolved. Most of the proteins would migrate on a diagonal line, however
proteins that got digested by the protease would drop under the diagonal. To validate this
method, we subjected 400 \ig of proteins from whole cell lysates to the diagonal gel in
the presence or absence of caspase-3. In the absence of caspase-3, all the proteins
migrated along the diagonal line. In the presence of caspase-3, its substrates were cleaved
and dropped below the diagonal (figure 5). Proteins were detected by staining of the
diagonal gel with Syproruby (Invitrogen) or by silver staining. We further validated this
technique by western blot. We loaded whole cell extracts from LPS pre-treated THP-1
cells on a 1st dimension gel and incubated the gel with caspase-1. The cleavage of the
known caspase-1 substrate IL-(3 was monitored by western analysis (figure 6). As shown
in figure 6, the 34 KDa pro-IL-ip was cleaved into the mature 17 KDa IL-ip by
caspase-1 and dropped under the diagonal.
We chose to work with human peripheral blood mononuclear cells (PBMCs) or with the
human monocytic cell-line THP-1 to screen for caspase-3, and caspase-1 substrates,
respectively. 400 fxg whole cell extracts were resolved on a 1st dimension SDS PAGE,
23
RESULTS AND DISCUSION
1. Caspases-1 and -3 substrates identified by the diagonal gel approach
The diagonal gel approach is a technique for protease substrate screening. It was recently
applied to identify the mitochondrial caspase-3 substrate NDUFS (Ricci et al., 2004).
Figure 4 schematically outlines this method. In general, whole cell lysates are separated
on a 1 st dimension SDS PAGE gel. The gellane is eut, dehydrated and then rehydrated in
a buffer containing the protease of interest. These steps wash out the SDS and allow the
protease to diffuse through the gel pores and digest its specifie substrates. Following this
in-gel digestion, the gellane is loaded horizontally on a second SDS-PAGE gel and the
proteins are resolved. Most of the proteins would migrate on a diagonal line, however
proteins that got digested by the protease would drop under the diagonal. To validate this
method, we subjected 400 !-tg of proteins from whole cell lysates to the diagonal gel in
the presence or absence of caspase-3. In the absence of caspase-3, aIl the proteins
migrated along the diagonalline. In the presence of caspase-3, its substrates were cleaved
and dropped below the diagonal (figure 5). Proteins were detected by staining of the
diagonal gel with Syproruby (Invitrogen) or by silver staining. We further validated this
technique by western blot. We loaded whole cell extracts from LPS pre-treated THP-l
cells on a 1 st dimension gel and incubated the gel with caspase-l. The cleavage of the
known caspase-l substrate IL-(3 was monitored by western analysis (figure 6). As shown
in figure 6, the 34 KDa pro-IL-lj3 was cleaved into the mature 17 KDa IL-lj3 by
caspase-l and dropped under the diagonal.
We chose to work with human peripheral blood mononuclear cells (PBMCs) or with the
human monocytic cell-line THP-l to screen for caspase-3, and caspase-l substrates,
respectively. 400 !-tg whole cell extracts were resolved on a 1 st dimension SDS PAGE,
23
the gel lanes were incubated in the presence or absence of 50ug recombinant caspase, and
then resolved again on a 2nd dimension SDS PAGE (figure 7). The proteins that dropped
under the diagonal were identified by mass spectrometry (at the McGill University and
Genome Quebec Innovation Centre using LC-MS). Using this approach, we identified 85
and 58 substrates for caspase-1 and - 3 , respectively (table 1). The criterion that we used
to identify a peptide from PMF (peptide mass fingerprint) is an ion score for each
peptide > 40, (which is a measure of the identity of the peptide obtained) and a peptide
coverage larger than 10% of the size of the caspase-1 cleavage product which was
extracted from under the diagonal. The identified substrates were classified into
subgroups related to their cellular function: cytoskeletal proteins, chaperons, nuclear
proteins, translation machinery proteins, immune proteins, glycolysis and bioenergetics
proteins, and proteins involved in signaling and trafficking (figure 8). Among the
identified substrates, 19 proteins were found to be known substrates of caspase-3 (Fischer
et al., 2003), for example, cytoskeletal proteins: actin, filamin, gelsolin, keratin and
vimentin; chaperons: Hsp70 and 90; proteins for RNA synthesis: hnRNP Al/A2/Cb;
translation machinery proteins: EEFA1, Nascent polypeptide-associated complex, etc.
(Fischer et al., 2003), which further confirmed accuracy and efficiency of this approach.
Interestingly, caspase-1 and -3 shared 25 common substrates, which accounted for
approximately 26% of total caspase-1 substrates and 38% of total caspase-3 substrates
identified by this method (figure 9A). The overlapping substrates were mostly
cytoskeletal proteins, accounting for 50% of the substrates in this subgroup (figure 9B).
Among the 5 common cytoskeletal substrates, 4 are known caspase-3 substrates cleaved
during apoptosis. The significant overlap of the substrates suggests that caspase-1, like
caspase-3, may execute cell death. In our substrate list, immune proteins were unique for
caspase-3. This may be due to the fact that we used PBMCs as a source of proteins in our
caspase-3 substrate screen. This suggested that caspase-3 cleaves common proteins as
well as cell-specific proteins, to fulfill its functions in apoptotic and non-apoptotic
24
the gellanes were incubated in the presence or absence of 50/-tg recombinant caspase, and
then resolved again on a 2nd dimension SDS PAGE (figure 7). The proteins that dropped
under the diagonal were identified by mass spectrometry (at the McGill University and
Genome Quebec Innovation Centre using LC-MS). Using this approach, we identified 85
and 58 substrates for caspase-1 and -3, respectively (table 1). The criterion that we used
to identify a peptide from PMF (peptide mass fingerprint) is an ion score for each
peptide> 40, (which is a measure of the identity of the peptide obtained) and a peptide
coverage larger than 10% of the size of the caspase-1 c1eavage product which was
extracted from under the diagonal. The identified substrates were c1assified into
subgroups related to their cellular function: cytoskeletal proteins, chaperons, nuc1ear
proteins, translation machinery proteins, immune proteins, glycolysis and bioenergetics
proteins, and proteins involved in signaling and trafficking (figure 8). Among the
identified substrates, 19 proteins were found to be known substrates of caspase-3 (Fischer
et al., 2003), for example, cytoskeletal proteins: actin, filamin, gelsolin, keratin and
vimentin; chaperons: Hsp70 and 90; proteins for RNA synthe sis: hnRNP AlIA2/Cb;
translation machinery proteins: EEFA1, Nascent polypeptide-associated complex, etc.
(Fischer et al., 2003), which further confirmed accuracy and efficiency of this approach.
Interestingly, caspase-1 and -3 shared 25 common substrates, which accounted for
approximately 26% of total caspase-1 substrates and 38% of total caspase-3 substrates
identified by this method (figure 9A). The overlapping substrates were mostly
cytoskeletal proteins, accounting for 50% of the substrates in this subgroup (figure 9B).
Among the 5 common cytoskeletal substrates, 4 are known caspase-3 substrates c1eaved
during apoptosis. The significant overlap of the substrates suggests that caspase-1, like
caspase-3, may execute cell death. In our substrate list, immune proteins were unique for
caspase-3. This may be due to the fact that we used PBMCs as a source ofproteins in our
caspase-3 substrate screen. This suggested that caspase-3 c1eaves common proteins as
weIl as cell-specific proteins, to fulfill its functions in apoptotic and non-apoptotic
24
processes. It is also worth noting that caspase-1 cleaved more proteins related to protein
translation, such as translation elongation factors and ribosomal proteins, than caspase-3
did, which implies that caspase-1 could have a dominant role in destroying the translation
machinery during cell death.
Although only few caspase-1 substrates are recorded in the literature, we identified a
large number of caspase-1 substrates in our screen. It was not surprising since we know
that the mature caspase-1 is a very active enzyme that cleaves pro-IL-l(3 very efficiently.
Moreover, caspase-1 is very similar to caspase-3 structurally and its active site also
recognizes the caspase-3 target motif, DEVD, with a binding affinity Kj = 17nM
(Nicholson and Thornberry, 1997). That might explain many common substrates obtained
for these two caspases. We validated eight substrates from our caspase-1 substrate list
using in vitro cleavage assay either by western blot of whole cell lysates or by in vitro
transcribed and translated S labeled substrates, two of them were not cleaved, six of
them were shown to be cleaved by caspase-1, which means that the chance of getting true
substrates from our list is 75%.
More recently, stimuli such as bacterial and viral components as well as some toxins have
been reported to activate caspase-1 via inflammasomes (Ogura et al., 2006). Our list of
caspase-1 substrates provides cues for catching in vivo cleavage of caspase-1 substrates
under those physiological conditions.
2. Caspase-1 targets in the glycolytic pathway
Glycolysis is the anaerobic catabolism of glucose, in which one molecule of glucose is
catabolized into two molecules of pyruvate and two ATP molecules. Under aerobic
condition, pyruvate is further oxidized by mitochondrial enzymes to CO2 and H2O
resulting in a higher yield of ATP. In the absence of oxygen, pyruvate is converted into
25
processes. It is also worth noting that caspase-l c1eaved more proteins related to prote in
translation, such as translation elongation factors and ribosomal proteins, than caspase-3
di d, which implies that caspase-l could have a dominant role in destroying the translation
machinery during cell death.
Although only few caspase-l substrates are recorded in the literature, we identified a
large number of caspase-l substrates in our screen. It was not surprising since we know
that the mature caspase-l is a very active enzyme that c1eaves pro-IL-IB very efficiently.
Moreover, caspase-l is very similar to caspase-3 structurally and its active site also
recognizes the caspase-3 target motif, DEVD, with a binding affinity Ki = l7nM
(Nicholson and Thornberry, 1997). That might explain many common substrates obtained
for these two caspases. We validated eight substrates from our caspase-l substrate list
using in vitro cleavage assay either by western blot of whole cell lysates or by in vitro
transcribed and translated 35S labeled substrates, two of them were not cleaved, six of
them were shown to be cleaved by caspase-l, which means that the chance of getting true
substrates from our list is 75%.
More recently, stimuli such as bacterial and viral components as well as sorne toxins have
been reported to activate caspase-l via inflammasomes (Ogura et al., 2006). Our list of
caspase-l substrates provides cues for catching in vivo cleavage of caspase-l substrates
under those physiological conditions.
2. Caspase-l targets in the glycolytic pathway
Glycolysis is the anaerobic catabolism of glucose, in which one molecule of glucose is
catabolized into two molecules of pyruvate and two ATP molecules. Under aerobic
condition, pyruvate is further oxidized by mitochondrial enzymes to CO2 and H20
resulting in a higher yield of ATP. In the absence of oxygen, pyruvate is converted into
25
lactate to regenerate NAD+ from NADH. Surprisingly, of all 11 enzymes involved in this
process, 6 were found to be targeted by caspase-1 in our diagonal gel experiment (figure
10). In addition, other enzymes related to glucose metabolism were also found in our
caspase-1 list including: aldose reductase, which is responsible for the conversion of
glucose to sorbitol, cAMP-dependent pyruvate kinase regulatory subunit alpha 2 for
glycogenolysis; fructose-1,6-bisphosphatase for gluconeogenesis; transaldolase and
N-acetylglucosamine kinase of the pentose pathway.
As a major energy source, glucose metabolism is essential for cellular functions. Severe
restrictions in glycolysis causes cell death, even in the presence of growth factors (Chi et
al., 2000). Importantly, death as a result of glucose limitation proceeds via apoptosis
(Vander Heiden et al., 2001). Cellular glucose metabolism is regulated by growth factors
(Summers and Birnbaum, 1997). Cellular transformation occurs when cell survival,
proliferation and metabolism become growth factor independent (Hanahan and Weinberg,
2000). Cancer cells develop strategies to shift their metabolism to glycolysis even in the
presence of normal oxygen pressure (Gatenby and Gillies, 2004). This phenomenon is
termed the Warburg effect (Warburg, 1930). The accelerated glycolysis confers cancer
cells with a selective advantage as it ensures ATP levels compatible with demands of fast
proliferation in hypoxic micro-environmental conditions. Changes in cellular metabolism
are sufficient to commit a cell to death or to more proliferation. Based on studies
demonstrating that glucose metabolism is implicated in cell death and survival, we have
reason to speculate that these two crucial processes, glycolysis and apoptosis, are linked.
Although caspase-1 is considered as an inflammatory caspase as its major function is the
maturation of IL-1J3 and IL-18 (Li et al., 1995), its role in cell death was also
demonstrated in certain types of cell, such as in macrophages (Fink and Cookson, 2005),
fibroblasts (Miura et al., 1993), neurons (Arai et al., 2006) and mammary epithelial cells
26
lactate to regenerate NAD+ from NADH. Surprisingly, of all Il enzymes involved in this
process, 6 were found to be targeted by caspase-1 in our diagonal gel experiment (figure
10). In addition, other enzymes related to glucose metabolism were also found in our
caspase-1 list including: aldose reductase, which is responsible for the conversion of
glucose to sorbitol, cAMP-dependent pyruvate kinase regulatory subunit alpha 2 for
glycogenolysis; fructose-1,6-bisphosphatase for gluconeogenesis; transaldolase and
N-acetylglucosamine kinase of the pentose pathway.
As a major energy source, glucose metabolism is essential for cellular functions. Severe
restrictions in glycolysis causes cell death, even in the presence of growth factors (Chi et
al., 2000). Importantly, death as a result of glucose limitation proceeds via apoptosis
(Vander Heiden et al., 2001). Cellular glucose metabolism is regulated by growth factors
(Summers and Birnbaum, 1997). Cellular transformation occurs when cell survival,
proliferation and metabolism become growth factor independent (Hanahan and Weinberg,
2000). Cancer cells develop strategies to shift their metabo1ism to glycolysis even in the
presence of normal oxygen pressure (Gatenby and Gillies, 2004). This phenomenon is
termed the Warburg effect (Warburg, 1930). The accelerated glycolysis conf ers cancer
cells with a selective advantage as it ensures ATP levels compatible with demands of fast
proliferation in hypoxic micro-environmental conditions. Changes in cellular metabolism
are sufficient to commit a cell to death or to more proliferation. Based on studies
demonstrating that glucose metabolism is implicated in cell death and survival, we have
reason to speculate that these two crucial processes, glycolylsis and apoptosis, are linked.
Although caspase-1 is considered as an inflammatory caspase as its major function is the
maturation of IL-ll3 and IL-18 (Li et al., 1995), its role in cell death was also
demonstrated in certain types of cell, such as in macrophages (Fink and Cookson, 2005),
fibroblasts (Miura et al., 1993), neurons (Arai et al., 2006) and mammary epithelial cells
26
(Boudreau et al., 1995). But the exact mechanism by which caspase-1 functions in cell
death is unclear. Since many glycolysis enzymes are targeted by caspase-1, investigation
of the cellular relevance of the cleavage of these substrates will help us to disclose how
caspase-1 executes cell death.
2.1 validation of the cleavage of the glycolysis enzymes by caspase-1
2.1.1 Validation of the caspase-1 cleaving glycolysis in vitro by western
blot
To validate the cleavage of the glycolysis enzymes by caspase-1, we performed in vitro
cleavage assays. In these experiments, THP-1 lysates were incubated with or without
recombinant caspase-1 then resolved by SDS PAGE on a 4-12% gel. The cleavage of
substrates was assessed by western blot using antibodies against aldolase, a-enolase, TIM,
GAPDH and IL-1(3. The cleavage assay of pyruvate kinase was not done due to lack of
an antibody. Cleavage of pro-IL-ip was chosen as a positive control. Aldolase migrated
as a doublet with one band running slightly faster than the full-length protein (figure 11).
Cleavage of a-enolase is obvious. The cleavage products are 37 KDa, 30 KDa and 20-25
KDa, suggesting more than one cleavage site may exist. Therefore the cleavage of
aldolase and a-enolase was confirmed by western blot, but that of TIM and GAPDH was
not detected by antibodies (figure 11).
Since caspase-1 and -3 share many common substrates, we further tested the cleavage of
a-enolase and aldolase with recombinant caspase-3. We incubated THP-1 lystates with
excessive amounts of recombinant caspase-3 (290 ng), but no cleavage was observed in
our western blot with antibodies against a-enolase and aldolase (figure 12). The activity
of the caspase-3 enzyme used (Sigma) was further tested with the selective caspase-3
27
(Boudreau et al., 1995). But the exact mechanism by which caspase-1 functions in cell
death is unc1ear. Since many glycolysis enzymes are targeted by caspase-1, investigation
of the cellular relevance of the cleavage of these substrates will help us to disclose how
caspase-1 executes cell death.
2.1 validation of the cleavage of the glycolysis enzymes by caspase-l
2.1.1 Validation of the caspase-l cleaving glycolysis in vitro by western
blot
To validate the c1eavage of the glycolysis enzymes by caspase-1, we performed in vitro
c1eavage assays. In these experiments, THP-1 lysates were incubated with or without
recombinant caspase-1 then resolved by SDS PAGE on a 4-12% gel. The c1eavage of
substrates was assessed by western blot using antibodies against aldolase, a-enolase, TIM,
GAPDH and IL-1f3. The c1eavage assay of pyruvate kinase was not done due to lack of
an antibody. Cleavage of pro-IL-1 13 was chosen as a positive control. Aldolase migrated
as a doublet with one band running slightly faster than the full-Iength prote in (figure 11).
Cleavage of a-enolase is obvious. The c1eavage products are 37 KDa, 30 KDa and 20-25
KDa, suggesting more than one cleavage site may exist. Therefore the cleavage of
aldolase and a-enolase was confirmed by western blot, but that of TIM and GAPDH was
not detected by antibodies (figure Il).
Since caspase-1 and -3 share manY common substrates, we further tested the c1eavage of
a-enolase and aldolase with recombinant caspase-3. We incubated THP-l lystates with
excessive amounts of recombinant caspase-3 (290 ng), but no cleavage was observed in
our western blot with antibodies against a-eno1ase and aldolase (figure 12). The activity
of the caspase-3 enzyme used (Sigma) was further tested with the selective caspase-3
27
fluorogenic peptide Ac-DEVD-AMC. Caspase-3 cleaved Ac-DEVD-AMC efficiently
with a rate of cleavage of 3.94 x 106 AFU (arbitrary fluorescent units), min"1. mg"1. We
concluded that caspase-1 cleaves a-enolase and aldolase, but caspase-3 does not.
2.1.2 Caspase-1 cleavage of in vitro transcribed and translated glycolysis
enzymes
To confirm that the glycolysis Enzymes were cleaved by caspase-1 directly, we
performed a second cleavage assay with 35S-methionine labeled in vitro transcribed and
translated (ITT) substrates.
To obtain the plasmids for in vitro transcription and translation, we first extracted mRNA
from THP-1 lysates, and cDNA of THP-1 was generated by reverse transcription,
a-enolase, aldolase, TIM and pyruvate kinase cDNA were cloned into pCDNA3.1, a T7
promoter containing vector. The GAPDH plasmid was purchased from ATCC and
re-cloned into pCDNA 3.1 vector for ITT. The primers designed for PCR are listed in
table 2. All plasmids used for ITT were confirmed by sequencing. Glycolysis enzyme
ITT products were produced using [35S] methionine and the TNT T7 coupled rabbit
reticulocyte lysate kit from Promega.
S labeled ITT glycolysis enzymes were incubated with or without recombinant
caspase-1 and -3 and then resolved by SDS PAGE. PARP, a substrate for both caspase-1
and -3 (Gu et al., 1995), was in vitro transcribed and translated and served as a positive
control. ITT products of a-enolase, aldolase, GAPDH, and TIM were cleaved by
caspase-1, but not by caspase-3; whileas pyruvate kinase was cleaved by both caspase-1
and -3 (figure 13). Caspase-1 strongly cleaved GAPDH, pyruvate kinase and a-enolase,
but aldolase cleavage was minor in this assay. To determine the cleavage efficiency, we
28
fluorogenic peptide Ac-DEVD-AMC. Caspase-3 cleaved Ac-DEVD-AMC efficiently
with a rate of cleavage of 3.94 x 106 AFU (arbitrary fluorescent units). min-l• mg- l
. We
concluded that caspase-1 cleaves a-enolase and aldolase, but caspase-3 do es not.
2.1.2 Caspase-l cleavage of in vitro transcribed and translated glycolysis
enzymes
To confirm that the glycolysis Enzymes were cleaved by caspase-1 directly, we
performed a second cleavage as say with 35S-methionine labeled in vitro transcribed and
translated (ITT) substrates.
To obtain the plasmids for in vitro transcription and translation, we first extracted mRNA
from THP-1 lysates, and cDNA of THP-1 was generated by reverse transcription.
a-enolase, aldolase, TIM and pyruvate kinase cDNA were cloned into pCDNA3.1, a T7
promoter containing vector. The GAPDH plasmid was purchased from ATCC and
re-cloned into pCDNA 3.1 vector for ITT. The primers designed for PCR are listed in
table 2. AH plasmids used for ITT were confirmed by sequencing. Glycolysis enzyme
ITT products were produced using e5S] methionine and the TNT T7 coupled rabbit
reticulocyte lysate kit from Promega.
35S labeled ITT glycolysis enzymes were incubated with or without recombinant
caspase-1 and -3 and then resolved by SDS PAGE. PARP, a substrate for both caspase-1
and -3 (Gu et al., 1995), was in vitro transcribed and translated and served as a positive
control. ITT products of a-enolase, aldolase, GAPDH, and TIM were cleaved by
caspase-1, but not by caspase-3; whileas pyruvate kinase was cleaved by both caspase-1
and -3 (figure 13). Caspase-1 strongly cleaved GAPDH, pyruvate kinase and a-enolase,
but aldolase cleavage was minor in this assay. To determine the cleavage efficiency, we
28
digested ITT glycolysis enzymes with increased amount of recombinant caspase-1 from
10 ng to 700 ng. The cleavage of GAPDH started to be seen with 10 ng of Caspase-1, but
at least 20 ng of caspase-1 was needed for the cleavage of a-enolase (figure 14). The
cleavage efficiency of GAPDH and a-enolase is in the same range as that of PARP,
which also needs a minimal 25 ng of caspase-1 for its cleavage in vitro (Gu et al., 1995).
However, the amount of caspase-1 needed for pro-IL-ip processing is 0.1 ng (figure 14).
So, caspase-1 cleaves pro-IL-ip much more efficiently. The PITSLRE kinase, a protein
related to the master mitotic protein kinase Cdc2, is another substrate for caspase-1.
Similar to pro-IL-l|3, it only requires 0.25 ng of caspase-1 for its cleavage in vitro
(Beyaert et al., 1997). Caspase-3, but not other caspases, also cleaves the PITSLRE
kinase at the same site used by caspase-1. However, caspase-3 cleaves the PITSLRE
kinase much less efficiently in that a minimum of 25 ng enzyme is required for the
cleavage of its ITT product (Beyaert et al., 1997). In vivo, a study with embryonic
fibroblasts from caspase-l"7" mice showed that the PITSLRE kinase was still cleaved to
the same extent in the caspase-1 deficient fibroblasts as compared to wild type cells, in
TNF-a induced apoptosis (Beyaert et al., 1997). Caspase-3 was presumably responsible
for the cleavage of the PITSLRE kinase in the caspase-1 deficient fibroblasts although it
cleaved this substrate less efficiently than caspase-1 in vitro. This demonstrates that
enzymes with lower efficiency in vitro could also have effects in vivo.
An interesting phenomenon was observed with enolase: in vitro transcription and
translation of full-length a-enolase resulted in the production of two polypeptides with
molecular weights of 48 kDa and 37 kDa, respectively (figure 14 A). The 48 KDa protein
is the expected size of full-length a-enolase. Further investigation disclosed that the 37
KDa protein is an alternative in-frame internal AUG translated isoform (Feo et al, 2000).
The 37 KDa alternative translation product was proposed to be MBP-1, the Myc
promoter-binding protein-1 that serves as a repressor of c-myc transcription (Ray and
29
digested ITT glycolysis enzymes with increased amount of recombinant caspase-1 from
10 ng to 700 ng. The cleavage of GAPDH started to be se en with 10 ng of Caspase-1, but
at least 20 ng of caspase-1 was needed for the cleavage of a-enolase (figure 14). The
cleavage efficiency of GAPDH and a-enolase is in the same range as that of PARP,
which also needs a minimal 25 ng of caspase-1 for its cleavage in vitro (Gu et al., 1995).
However, the amount of caspase-1 needed for pro-IL-1~ processing is 0.1 ng (figure 14).
So, caspase-1 cleaves pro-IL-1~ much more efficiently. The PITSLRE kinase, a protein
related to the master mitotic prote in kinase Cdc2, is another substrate for caspase-1.
Similar to pro-IL-l[3, it only requires 0.25 ng of caspase-l for its cleavage in vitro
(Beyaert et al., 1997). Caspase-3, but not other caspases, also cleaves the PITSLRE
kinase at the same site used by caspase-1. However, caspase-3 cleaves the PITSLRE
kinase much less efficiently in that a minimum of 25 ng enzyme is required for the
cleavage of its ITT product (Beyaert et al., 1997). In vivo, a study with embryonic
fibroblasts from caspase-1-1- mice showed that the PITSLRE kinase was still cleaved to
the same extent in the caspase-1 deficient fibroblasts as compared to wild type cells, in
TNF-a induced apoptosis (Beyaert et al., 1997). Caspase-3 was presumably responsible
for the cleavage of the PITSLRE kinase in the caspase-1 deficient fibroblasts although it
cleaved this substrate less efficiently than caspase-1 in vitro. This demonstrates that
enzymes with lower efficiency in vitro could also have effects in vivo.
An interesting phenomenon was observed with enolase: in vitro transcription and
translation of full-Iength a-enolase resulted in the production of two polypeptides with
molecular weights of 48 kDa and 37 kDa, respectively (figure 14 A). The 48 KDa protein
is the expected size of full-Iength a-enolase. Further investigation disclosed that the 37
KDa protein is an alternative in-frame internaI AUG translated isoform (Feo et al., 2000).
The 37 KDa alternative translation product was proposed to be MBP-1, the Myc
promoter-binding protein-l that serves as a repressor of c-myc transcription (Ray and
29
Miller, 1991). The evidence to support this is: First, the MBP-1 cDNA shares 97%
similarity with the cDNA encoding a-enolase, both in the coding region and in the
3'-untranslated sequence (Giallongo et al , 1986). Second, the chromosomal location of
MBP-1 on human chromosome lp36, is in close proximity to that of the gene encoding
a-enolase (Onyango et al., 1998). Third, the 37 kDa alternatively translated isoform is
preferentially localized in the nucleus, sharing with MBP-1 the capability to
downregulate the transcription of c-myc (Feo et al., 2000). The antibody against
a-enolase detected both the 48 KDa and 37 KDa proteins in many cell lines (Feo et al.,
2000), but the antibody that we used (from Santa Cruz) only detected the 48 KDa form in
THP-1 cell lysate (figure 11), whereas a 37 KDa cleavage fragment was detected in the
lane digested with caspase-1 by western blot (figure 11). Comparison of a-enolase's
western blot and ITT gel revealed that the cleavage product of around 20-25 KDa
fragment was detected by both methods. The 37 KDa cleavage fragment, which was
observed by western blot, could also exist on the ITT gel, however it would be masked by
the 37 KDa alternative translation product (figure 14 A). It should be noted that caspase-1
cleaved both forms of a-enolase, the 48 KDa and 37 KDa proteins. Further work needs to
be done to examine whether caspase-1 cleaves a-enolase to generate a 37 KDa fragment.
This could be accomplished by blocking the internal translation of the 37 KDa isoform.
Since internal translation of the 37 KDa isoform starts at Met97 in the a-enolase cDNA
(Feo et al., 2000), point mutation of this site could possibly block internal translation. If
caspase-1 cleaved a-enolase into a 37 KDa fragment, it would be interesting to examine
whether this cleavage product would have similar function as MBP-1.
ITT of aldolase also showed a minor alternative translation form of around 22 KDa
(figure 14 B), but this alternative translation form is not recorded in the literature.
Caspase-1 cleaves both translation products, and the cleavage fragments migrate at
around 28 KDa and 10 -15 KDa.
30
Miller, 1991). The evidence to support this is: First, the MBP-l cDNA shares 97%
similarity with the cDNA encoding a-enolase, both in the coding region and in the
3'-untranslated sequence (Giallongo et al., 1986). Second, the chromosomallocation of
MBP-l on human chromosome 1 p36, is in close proximity to that of the gene encoding
a-enolase (Onyango et al., 1998). Third, the 37 kDa alternatively translated isoform is
preferentially localized in the nucleus, sharing with MBP-l the capability to
downregulate the transcription of c-myc (Feo et al., 2000). The antibody against
a-enolase detected both the 48 KDa and 37 KDa proteins in many celllines (Feo et al.,
2000), but the antibody that we used (from Santa Cruz) only detected the 48 KDa form in
THP-l celllysate (figure 11), whereas a 37 KDa cleavage fragment was detected in the
lane digested with caspase-l by western blot (figure 11). Comparison of a-enolase's
western blot and ITT gel revealed that the cleavage product of around 20-25 KDa
fragment was detected by both methods. The 37 KDa cleavage fragment, which was
observed by western blot, could also exist on the ITT gel, however it would be masked by
the 37 KDa alternative translation product (figure 14 A). It should be noted that caspase-l
cleaved both forms of a-enolase, the 48 KDa and 37 KDa proteins. Further work needs to
be done to examine whether caspase-l cleaves a-enolase to generate a 37 KDa fragment.
This could be accomplished by blocking the internal translation of the 37 KDa isoform.
Since internaI translation of the 37 KDa isoform starts at Meë7 in the a-enolase cDNA
(Feo et al., 2000), point mutation of this site could possibly block internaI translation. If
caspase-l cleaved a-enolase into a 37 KDa fragment, it would be interesting to examine
whether this cleavage product would have similar function as MBP-l.
ITT of aldolase also showed a minor alternative translation form of around 22 KDa
(figure 14 B), but this alternative translation form is not recorded in the literature.
Caspase-l cleaves both translation products, and the cleavage fragments migrate at
around 28 KDa and 10 - 15 KDa.
30
2.2 Identification of the caspase-1 cleavage site in the glycolysis Enzymes
After we have confirmed that the glycolysis enzymes were directly cleaved by caspase-1,
we then sought to map the caspase-1 cleavage sites. As caspases have a near absolute
preference for Asp residues in the PI position, we scanned the amino acid sequence in
cc-enolase and GAPDH for possible caspase cleavage motifs, which would generate the
fragments shown in the ITT cleavage experiments (Figure 14). We used site-directed
mutagenesis to change the possible Aspartate residues to Alanine in four possible
positions in GAPDH and a-enolase (figure 15A, 16A). As shown in figure 15C, GAPDH
mutant D189A was resistant to caspase-1 cleavage, whereas all four mutations in
a-enolase couldn't block its cleavage by caspase-1 (figure 16 B). Inspection of the
cleavage site of GAPDH from several species revealed that the cleavage site from PI -
P4 and PI ' (KTVD189 I G) are completely conserved among these species (figure 15 B).
We have therefore mapped D189 as a single caspase-1 cleavage site in GAPDH.
2.3 In vivo studies on the cleavage of the glycolysis enzymes upon
activation of Caspase-1
After we have identified that those glycolysis enzymes were caspase-1 substrates in vitro,
we then tested if these substrates were cleaved in vivo in response to caspase-1 activation.
2.3.1 Activation of caspase-1 by transfection of procaspase-1 into HEK293
cells
Several studies have shown that caspase-1 cleaves its substrates when over-expressed
into HEK293 (Kahns et al., 2003; Lamkanfi et al., 2004; Zhang et al , 2003a). Since the
glycolysis enzymes are constitutively expressed in HEK293, we transfected procaspase-1
into HEK293, but auto-processing of caspase-1 was not observed by western blot, and
31
2.2 Identification of the caspase-1 cleavage site in the glycolysis Enzymes
After we have confirmed that the glycolysis enzymes were directly cleaved by caspase-I,
we then sought to map the caspase-I cleavage sites. As caspases have a near absolute
preference for Asp residues in the Pl position, we scanned the amino acid sequence in
a-enolase and GAPDH for possible caspase cleavage motifs, which would generate the
fragments shown in the ITT cleavage experiments (Figure 14). We used site-directed
mutagenesis to change the possible Aspartate residues to Alanine in four possible
positions in GAPDH and a-enolase (figure I5A, I6A). As shown in figure I5e, GAPDH
mutant D189 A was resistant to caspase-l cleavage, whereas ail four mutations in
a-enolase couldn't block its cleavage by caspase-l (figure 16 B). Inspection of the
cleavage site of GAPDH from several species revealed that the cleavage site from Pl -
P4 and Pl' (KTVD189 + G) are completely conserved among these species (figure 15 B).
We have therefore mapped D189 as a single caspase-I cleavage site in GAPDH.
2.3 In vivo studies on the cleavage of the glycolysis enzymes upon
activation ofCaspase-1
After we have identified that those glycolysis enzymes were caspase-l substrates in vitro,
we then tested if these substrates were cleaved in vivo in response to caspase-l activation.
2.3.1 Activation of caspase-1 by transfection ofprocaspase-1 into HEK293
cells
Several studies have shown that caspase-l cleaves its substrates when over-expressed
into HEK293 (Kahns et al., 2003; Lamkanfi et al., 2004; Zhang et al., 2003a). Since the
glycolysis enzymes are constitutively expressed in HEK293, we transfected procaspase-l
into HEK293, but auto-processing of caspase-l was not observed by western blot, and
31
a-enolase and aldolase were not cleaved (data not shown).
2.3.2 Activation ofcaspase-1 in THP-1 cells treated with LPS plus ATP or
Nigericin
Caspase-1 is constitutively expressed in monocytes and macrophages (Lin et al., 2000).
Previous research has shown that activation of caspase-1 in macrophages occurs in
response to Toll-like receptor activation and ATP treatment, the latter activating the
P2X7 receptor which decreases intracellular K+ levels by forming a pore in the plasma
membrane (Perregaux and Gabel, 1994; Surprenant et al., 1996). The toxin nigericin, a
potassium ionophore was also found to activate caspase-1 and leads to the release of
IL-ip by macrophages (Perregaux and Gabel, 1994). This process is dependent on
caspase-1 activation within the NALP3 inflammasome (Mariathasan et al., 2006). We
have applied this activation model to THP-1 cells. In our experiments, we pre-treated
THP-1 cells with PMA to differentiate the cells into macrophages, and then treated with
LPS and ATP or Nigericin. Processing of Pro-IL-ip into the 17 KDa form was shown on
western blot (figure 17). Activated caspase-1 (plO) was not detected in western blot. The
possible reason is that the antibody used in these experiments was raised against mouse
caspase-1 and is not sensitive to human caspase-1. But the cleavage of a-enolase and
aldolase was not seen by western blot. One possibility is that the activation ofcaspase-1
was not robust enough to cleave these substrates.
2.3.3 Activation ofcaspase-1 in Salmonella infected THP-1 cells
The Gram-negative bacteria Salmonella was reported to infect macrophages and induce
cell death in the host cells by activating caspase-1 (Hersh et al., 1999). Cell death of host
cells can be advantageous or detrimental to pathogenesis. In the case of Salmonella, the
32
a-enolase and aldolase were not cleaved (data not shown).
2.3.2 Activation of caspase-l in THP-l cells treated with LPS plus ATP or
Nigericin
Caspase-1 is constitutively expressed in monocytes and macrophages (Lin et al., 2000).
Previous research has shown that activation of caspase-1 in macrophages occurs in
response to Toll-like receptor activation and ATP treatment, the latter activating the
P2X7 receptor which decreases intracellular K+ levels by forming a pore in the plasma
membrane (Perregaux and Gabel, 1994; Surprenant et al., 1996). The toxin nigericin, a
potassium ionophore was also found to activate caspase-1 and leads to the release of
IL-1~ by macrophages (Perregaux and Gabel, 1994). This process is dependent on
caspase-1 activation within the NALP3 inflammasome (Mariathasan et al., 2006). We
have applied this activation model to THP-1 cells. In our experiments, we pre-treated
THP-1 cells with PMA to differentiate the cells into macrophages, and then treated with
LPS and ATP or Nigericin. Processing ofPro-IL-1~ into the 17 KDa form was shown on
western blot (figure 17). Activated caspase-1 (plO) was not detected in western blot. The
possible reason is that the antibody used in these experiments was raised against mouse
caspase-1 and is not sensitive to human caspase-1. But the cleavage of a-enolase and
aldolase was not seen by western blot. One possibility is that the activation of caspase-1
was not robust enough to c1eave these substrates.
2.3.3 Activation of caspase-l in Salmonella infected THP-l cells
The Gram-negative bacteria Salmonella was reported to infect macrophages and induce
cell death in the host cells by activating caspase-1 (Hersh et al., 1999). Cell death of host
cells can be advantageous or detrimental to pathogenesis. In the case of Salmonella, the
32
bacteria induces cell death as a virulence strategy for its systematic spreading into the
host (Monack et al., 2000). The mechanism of caspase-1 activation involves the assembly
of an IPAF inflammasome (Mariathasan et al., 2006), and the consequent activation of
the ASC pyroptosome (Alnemri, 2007). Salmonella infection-induced pyroptosis has
been characterized as a caspase-1 dependent cell death as it is distinct from classical
apoptosis and caspase-3 is not involved in this process (Monack et al., 2001). Therefore it
was very important to investigate caspase-1 substrates cleavage during this process. We
infected THP-1 cells with Salmonella. We first treated THP-1 cells with PMA to
differentiate the cells into macrophages, primed the cells with a low dose of LPS, and
then infected with Salmonella at different multiplicity of infection (moi). 24 hours
post-infection, massive macrophage cell death was observed in culture. In this
experiment, activation of caspse-1 was monitored using the fluorescent reagent
FLICAcaspl, which binds to the active form of caspase-1 with high affinity. Our results
showed that PMA treatment induced around 10% caspase-1 activation detected by
FLICAeaspl (figure 18), however, no pro-IL-ip processing or caspase-1 auto-cleavage
were observed in response to PMA. In response to LPS treatment, caspase-1 activation
increased to 31%, but pro-IL-l|3 and caspase-1 were not yet processed. Salmonella
infection activated caspase-1 up to nearly 100% and pro-IL-ip was processed into IL-1(3.
Protein level of caspase-1 is low in western blot, which is consistent with previous
research showing that caspase-1 is secreted with IL-ip from the macrophage upon
activation (Andrei et al., 2004). Surprisingly, cc-enolase and aldolase were completely
degraded (The bands detected by western blot were due to cross reactivity of the
antibodies with bacterial enolase, which migrated slightly faster than mammalian
a-enolase derived from THP-1). This suggests that they might be degraded in the cell
after a 24 hr-infection with Salmonella. It has been reported that caspase substrates
DIAP1 (an Drosophila inhibitor of apoptosis) and TWIST (a transcription factor essential
for mesodermal development) are degraded by ubiquitin/proteosome pathways after
33
bacteria induces cell death as a virulence strategy for its systematic spreading into the
host (Monack et al., 2000). The mechanism of caspase-l activation involves the assembly
of an IPAF inflammasome (Mariathasan et al., 2006), and the consequent activation of
the ASC pyroptosome (Alnemri, 2007). Salmonella infection-induced pyroptosis has
been characterized as a caspase-l dependent cell death as it is distinct from classical
apoptosis and caspase-3 is not involved in this process (Monack et al., 2001). Therefore it
was very important to investigate caspase-l substrates cleavage during this process. We
infected THP-l cells with Salmonella. We first treated THP-l cells with PMA to
differentiate the cells into macrophages, primed the cells with a low dose of LPS, and
then infected with Salmonella at different multiplicity of infection (moi). 24 ho urs
post-infection, massive macrophage cell death was observed in culture. In this
experiment, activation of caspse-l was monitored using the fluorescent reagent
FUCA casPI, which binds to the active form of caspase-l with high affinity. Our results
showed that PMA treatment induced around 10% caspase-l activation detected by
FUCA caspl (figure 18), however, no pro-IL-l ~ processing or caspase-l auto-cleavage
were observed in response to PMA. In response to LPS treatment, caspase-l activation
increased to 31 %, but pro-IL-l ~ and caspase-l were not yet processed. Salmonella
infection activated caspase-l up to nearly 100% and pro-IL-l ~ was processed into IL-l~.
Protein level of caspase-l is low in western blot, which is consistent with previous
research showing that caspase-l is secreted with IL-l ~ from the macrophage upon
activation (Andrei et al., 2004). Surprisingly, a-enolase and aldolase were completely
degraded (The bands detected by western blot were due to cross reactivity of the
antibodies with bacterial enolase, which migrated slightly faster than mammalian
a-enolase derived from THP-l). This suggests that they might be degraded in the cell
after a 24 hr-infection with Salmonella. It has been reported that caspase substrates
DIAPI (an Drosophila inhibitor ofapoptosis) and TWIST (a transcription factor essential
for mesodermal development) are degraded by ubiquitin/proteosome pathways after
33
caspase cleavage (Demontis et al., 2006; Ditzel et al., 2003). In the case of DIAP1, when
it is cleaved by a caspase, the N-end Asn is exposed, which sensitizes the protein to be
degraded by the ubiquitin dependent N-end rule pathway (Ditzel et al., 2003). Similarly,
caspase cleaves off the C-terminus of TWIST, which greatly affects stability of the
protein and thus triggers its degradation by the lysine dependent ubiquitination pathway
(Demontis et al., 2006). Based on the depletion of the glycolysis enzymes after
Salmonella infection in THP-1 cells and on the results showing that the glycolysis
enzymes were cleaved by caspase-1 in vitro, we hypothesize that the caspase-1 cleavage
might target the glycolysis enzymes to ubiquitin-dependent degradation.
2.3.4 Examination of the glycolysis enzymes cleavage in the mice muscle
in Septic shock
Septic shock is characterized by a systemic inflammatory response to an infection or
injury, in which a group of inflammatory cytokines, such as TNFcc, IL-ip, IL-6, IL-8 and
IL-18 are released by inflammatory cells (Cavaillon et al., 2003; Endo et al., 2000;
Grobmyer et al., 2000). Excessive activation of caspase-1 has been reported in this
condition (Oberholzer et al., 2000). As we know, glucose metabolism is linked to muscle
function in that ATP provides energy for muscle contraction. Therefore, Glucose
metabolism, as the major source of energy in the skeletal muscle, is essential for muscle
function. Patients in septic shock develop muscle weakness caused by loss of
contractile ability of their skeletal muscles (Ginz et al., 2005). Posttranslational
modifications of glycolysis enzymes have been observed in the diaphragm of septic
rodents (Barreiro et al., 2005; Hussain et al., 2006). LPS injection in rats has been shown
to cause muscle wasting: The isometric force generated by the diaphragm significantly
and progressively declined in this septic shock model (Barreiro et al., 2005). We
examined the cleavage of the glycolysis enzymes in the diaphragm of mice subjected to
the septic shock model. For this part of the work, we collaborated with Dr. Sabah
34
caspase cleavage (Demontis et al., 2006; Ditzel et al., 2003). In the case of DIAP l, when
it is cleaved by a caspase, the N-end Asn is exposed, which sensitizes the protein to be
degraded by the ubiquitin dependent N-end rule pathway (Ditzel et al., 2003). Similarly,
caspase cleaves off the C-terminus of TWIST, which greatly affects stability of the
protein and thus triggers its degradation by the lysine dependent ubiquitination pathway
(Demontis et al., 2006). Based on the depletion of the glycolysis enzymes after
Salmonella infection in THP-l cells and on the results showing that the glycolysis
enzymes were cleaved by caspase-l in vitro, we hypothesize that the caspase-l cleavage
might target the glycolysis enzymes to ubiquitin-dependent degradation.
2.3.4 Examination of the glyeolysis enzymes cleavage in the miee muscle
in Septie shoek
Septic shock is characterized by a systemic inflammatory response to an infection or
injury, in which a group of inflammatory cytokines, such as TNF a, IL-l (3, IL-6, IL-8 and
IL-l8 are released by inflammatory cells (Cavaillon et al., 2003; Endo et al., 2000;
Grobmyer et al., 2000). Excessive activation of caspase-l has been reported in this
condition (Oberholzer et al., 2000). As we know, glucose metabolism is linked to muscle
function in that ATP provides energy for muscle contraction. Therefore, Glucose
metabolism, as the major source of energy in the skeletal muscle, is essential for muscle
function. Patients in septic shock develop muscle weakness caused by loss of
contractile ability of their skeletal muscles (Ginz et al., 2005). Posttranslational
modifications of glycolysis enzymes have been observed in the diaphragm of septic
rodents (Barreiro et al., 2005; Hussain et al., 2006). LPS injection in rats has been shown
to cause muscle wasting: The isometric force generated by the diaphragm significantly
and progressively declined in this septic shock model (Barreiro et al., 2005). We
examined the cleavage of the glycolysis enzymes in the diaphragm of mice subjected to
the septic shock model. For this part of the work, we collaborated with Dr. Sabah
34
Hussain's lab. Mice were injected intraperitoneally with LPS and killed 12 hrs or 24 hrs
after injection. The diaphragms were excised to extract protein. 50 ug of total proteins
were subjected SDS PAGE and probed with antibodies against a-enolase, adolase,
GAPDH, TIM and actin. Actin was chosen as a loading control. Our results show that
proforms of all the glycolysis enzymes examined were greatly decreased 24 hrs post LPS
injection (figure 19). Activation of caspase-1 was confirmed by the detection of
autocatalytic processing of caspase-1 into the pi0 and p20 subunits in the 24 hrs samples
(figure 19). ProIL-l(3 was induced 24 hrs after LPS injection, but the cleaved IL-lp was
not seen due to its secretion. These results correlate with our previous results with
Salmonella infection, in which a-enolase and aldolase were degraded 24 hrs
post-infection (figure 18). The consequence of the degradation of the glycolysis enzymes
would be to shut down the glycolytic energy source, leading to cell death. In future
experiments, we will investigate the degradation of the glycolysis enzymes in caspase-1
knock-out mice upon LPS injection and compare the muscle contractile ability from wild
type and caspase-1 knock-out mice during septic shock.
2.3.5 The Glycolytic rate is reduced during Salmonella infection ofTHP-1
cells
To test our hypothesis that the consequence of the cleavage of the glycolysis enzymes is
to shut down this energy source and therefore promote cell death, we compared the
change in the glycolytic rate of THP-1 cells with and without Salmonella infection. In
this experiment, we pre-treated THP-1 cells with PMA and infected with salmonella for 4
hrs. The glycolytic rate of THP-1 cells was assessed by measuring lactate production in
the presence or absence of the caspase-1 inhibitor YVAD-FMK. As shown in figure 20,
lactate production decreased by 2 fold after THP-1 cells were infected with Salmonella,
and caspase-1 inhibitor YVAD-FMK partially reversed this effect. Our results have
35
I~ Hussain's labo Mice were injected intraperitoneally with LPS and killed 12 hrs or 24 hrs
after injection. The diaphragms were excised to extract prote in. 50 Ilg of total proteins
were subjected SDS PAGE and probed with antibodies against a-enolase, adolase,
GAPDH, TIM and actin. Actin was chosen as a loading control. Our results show that
proforms of all the glycolysis enzymes examined were greatly decreased 24 hrs post LPS
injection (figure 19). Activation of caspase-l was confirmed by the detection of
autocatalytic processing of caspase-l into the plO and p20 subunits in the 24 hrs samples
(figure 19). ProIL-l~ was induced 24 hrs after LPS injection, but the cleaved IL-l~ was
not seen due to its secretion. These results correlate with our previous results with
Salmonella infection, in which a-enolase and aldolase were degraded 24 hrs
post-infection (figure 18). The consequence of the degradation of the glycolysis enzymes
would be to shut down the glycolytic energy source, leading to cell death. In future
experiments, we will investigate the degradation of the glycolysis enzymes in caspase-l
knock-out mice upon LPS injection and compare the muscle contractile ability from wild
type and caspase-l knock-out mice during septic shock.
2.3.5 The Glycolytic rate is reduced during Salmonella infection of THP-l
cells
To test our hypothesis that the consequence of the c1eavage of the glycolysis enzymes is
to shut down this energy source and therefore promote cell death, we compared the
change in the glycolytic rate of THP-l cells with and without Salmonella infection. In
this experiment, we pre-treated THP-l cells with PMA and infected with salmonella for 4
hrs. The glycolytic rate of THP-l cells was assessed by measuring lactate production in
the presence or absence of the caspase-l inhibitor YVAD-FMK. As shown in figure 20,
lactate production decreased by 2 fold after THP-l cells were infected with Salmonella,
and caspase-l inhibitor YVAD-FMK partially reversed this effect. Our results have
35
demonstrated that glycolysis is impaired in THP-1 cells infected with Salmonella, and
that caspase-1 is involved in this process.
r^
36
demonstrated that glycolysis is impaired in THP-l cells infected with Salmonella, and
that caspase-l is involved in this process.
36
CONCLUSIONS AND FUTURE DIRECTIONS:
We have applied a proteomic approach, the diagnonal gel method, to screen for caspase
substrates. This method is based on in-gel digestion of substrates by caspases and allows
identification of caspase substrates from whole cell extracts. In our experiments, we have
identified 85 and 58 potential substrates for caspases-1 and - 3 , respectively. We have
validated 8 selected caspase-1 substrates using in vitro cleavage assays. Among the 58
caspase-3 substrates that we obtained, 19 substrates have been reported in the literature as
bona fide caspase-3 substrates. Our results have also demonstrated that caspases-1 and -3
share 25 common substrates, which account for approximately 26% of total caspase-1
substrates and 38% of total caspase-3 substrates identified by this method. Those
caspase-1 and -3 substrates are implicated in basic cellular functioning and survival
(maintenance of the cytoskeleton, translation, glycolysis, bioenergetics, signaling and
trafficking).
Another proteomic method, the two-dimensional gel electrophoresis (2D), has been
applied to search for caspase substrates. In this method, proteins derived from healthy or
apoptotic cells were separated using 2D gels and the migration patterns of proteins from a
healthy versus apoptotic cell were compared (Gerner et al., 2000). Although this method
is also efficient, as few hundred altered protein spots were detected on the 2D gels
(Gerner et al., 2000), it has the limitation that many substrates of other proteases were
also detected. However, if we combine the results from the diagonal gel and 2D gel
approaches, the overlapping substrates from these two methods would represent potential
true in vivo caspase substrates. In addition, one advantage of our in vitro screening is that
it could uncover substrates that are undergone degradation soon after their cleavage by
caspases in vivo. Since those substrates are quickly degraded upon apoptotic stimulation,
it is impossible to catch them with in vivo screening methods.
37
CONCLUSIONS AND FUTURE DIRECTIONS:
We have applied a proteomic approach, the diagnonal gel method, to screen for caspase
substrates. This method is based on in-gel digestion of substrates by caspases and allows
identification of caspase substrates from whole cell extracts. In our experiments, we have
identified 85 and 58 potential substrates for caspases-l and -3, respectively. We have
validated 8 selected caspase-l substrates using in vitro cleavage assays. Among the 58
caspase-3 substrates that we obtained, 19 substrates have been reported in the literature as
bona fide caspase-3 substrates. Our results have also demonstrated that caspases-l and-3
share 25 common substrates, which account for approximately 26% of total caspase-l
substrates and 38% of total caspase-3 substrates identified by this method. Those
caspase-l and -3 substrates are implicated in basic cellular functioning and survival
(maintenance of the cytoskeleton, translation, glycolysis, bioenergetics, signaling and
trafficking).
Another proteomic method, the two-dimensional gel electrophoresis (2D), has been
applied to search for caspase substrates. In this method, proteins derived from healthy or
apoptotic cells were separated using 2D gels and the migration patterns of proteins from a
healthy versus apoptotic cell were compared (Gerner et al., 2000). Although this method
is also efficient, as few hundred altered protein spots were detected on the 2D gels
(Gerner et al., 2000), it has the limitation that many substrates of other proteases were
also detected. However, if we combine the results from the diagonal gel and 2D gel
approaches, the overlapping substrates from these two methods would represent potential
true in vivo caspase substrates. In addition, one advantage of our in vitro screening is that
it could uncover substrates that are undergone degradation soon after their cleavage by
caspases in vivo. Since those substrates are quickly degraded upon apoptotic stimulation,
it is impossible to catch them with in vivo screening methods.
37
Our data have indicated that the glycolysis pathway is targeted by caspase-1. Five
glycolysis enzymes, a-enolase, aldolase, TIM, GAPDH and pyruvate kinase, are cleaved
by caspase-1. The processing of the above glycolysis enzymes by caspase-1 has been
confirmed by incubating nanogram amounts of purified recombinant human caspase-1
with S-methionine-labelled in vitro transcribed and translated substrates. Additionally,
we have identified the caspase-1 cleavage site in GAPDH as aspartate 189. GAPDH
with a point mutation of Aspl89->Ala (D189-A) is resistant to cleavage by caspase-1 in
vitro. In vivo cleavage as well as the consequence of this cleavage remains to be
investigated.
We have examined the in vivo cleavage of the glycolysis enzymes in three caspase-1
activation systems: 1) Stimulation of THP-1 cells with LPS plus ATP or nigericin. This
condition has been shown to activate caspase-1 within the NALP-3 inflammasome and
results in the processing and secretion of the caspase-1 substrate IL-ip. In this system, we
didn't detect any change in the glycolysis enzyme levels or processing. 2) Salmonella
typhimurium infection of THP-1 cells. This condition has been reported to activate
caspase-1 within an IPAF inflammasome. In both conditions we failed to observe
caspase-1 activation and processing by western blot, but we detected high FLICAcasp-1
staining in response to 24 hour-Salmonella infection, at which point the cells were in an
advanced phase of cell death and glycolysis enzymes that we examined were degraded. 3)
Septic shock mice model with LPS induced endotoximia. In this condition,
auto-processing of caspase-1 was detected and the protein level of the glycolysis enzymes,
including a-enolase, aldolse, GAPDH and TIM were greatly decreased after 24 hr LPS
injection although cleavage products of these substrates were not observed. This result
correlates with that we obtained in Salmonella infection. In combination with our results
of the cleavage of the glycolysis enzymes by caspase-1 in vitro, we can speculate that
38
Our data have indicated that the glycolysis pathway is targeted by caspase-1. Five
glycolysis enzymes, a-enolase, aldolase, TIM, GAPDH and pyruvate kinase, are cleaved
by caspase-l. The processing of the above glycolysis enzymes by caspase-1 has been
confirmed by incubating nanogram amounts of purified recombinant human caspase-1
with 35S-methionine-Iabelled in vitro transcribed and translated substrates. Additionally,
we have identified the caspase-1 cleavage site in GAPDH as aspartate 189. GAPDH
with a point mutation of Asp 189~ Ala (D189 -A) is resistant to cleavage by caspase-1 in
vitro. In vivo cleavage as well as the consequence of this cleavage remains to be
investigated.
We have examined the in vivo cleavage of the glycolysis enzymes in three caspase-1
activation systems: 1) Stimulation of THP-1 ceIls with LPS plus ATP or nigericin. This
condition has been shown to activate caspase-1 within the NALP-3 inflammasome and
results in the processing and secretion of the caspase-l substrate IL-1 13. In this system, we
didn't detect any change in the glycolysis enzyme levels or processing. 2) Salmonella
typhimurium infection of THP-1 cells. This condition has been reported to activate
caspase-1 within an IPAF inflammasome. In both conditions we failed to observe
caspase-1 activation and processing by western blot, but we detected high FLICAcasp-l
staining in response to 24 hour-Salmonella infection, at which point the cells were in an
advanced phase of ceIl death and glycolysis enzymes that we examined were degraded. 3)
Septic shock mice model with LPS induced endotoximia. In this condition,
auto-processing of caspase-1 was detected and the protein level of the glycolysis enzymes,
including a-enolase, aldolse, GAPDH and TIM were greatly decreased after 24 hr LPS
injection although cleavage products of these substrates were not observed. This result
correlates with that we obtained in Salmonella infection. In combination with our results
of the cleavage of the glycolysis enzymes by caspase-l in vitro, we can speculate that
38
those glycolysis enzymes could be degraded after cleavage by caspase-1 in the above two
conditions. Indeed, caspase substrates are reported to be degraded after cleavage
(Demontis et al., 2006; Ditzel et al, 2003; Du et al., 2005). Caspases initiate degradation
of their substrates and target them to be recognized by the ubiquitin/proteosome system.
Accumulating evidences link the ubiquitin/proteosome pathway to caspase function and
many researchers have implied a function of caspase-3 in the degradation of proteins
under pathogenic conditions (Mitch, 2007; Ottenheijm et al., 2006). The involvement of
caspase-1 in the degradation events has not been previously reported.
In vivo cleavage of certain substrates by caspases is cell type specific and
stimulus-specific. For example, GATA-1 is cleaved by caspases in erythroid cells
undergoing apoptosis in response to erythropeitin (EPO) deprivation or death receptor
stimulation ((De Maria et al., 1999), but remains uncleaved in erythroid cells undergoing
terminal differentiation (Zermati et al., 2001); actin can be cleaved by caspases in U397
leukemia cells, neurons and thymocytes, but not in other cell types undergoing apoptosis
(Earnshaw et al., 1999). So, this raises the difficulty of capturing the in vivo cleavage
event of a known in vitro caspase substrate.
In addition to the above-described caspase-1 activation systems, few other caspase-1
activation systems remain to be tested and in which we could examine the cleavage of the
glycolysis enzymes. Caspase-1 knockout mice develop normally and appear healthy and
fertile, but thymocytes from caspase-1 deficient mice are more resistant to apoptosis
induced by anti-CD95 antibody (Kuida et al , 1995; Li et al., 1995). This suggests a
non-redundant function of caspase-1 in this cell type. It would be worthwhile to explore
the cleavage of the glycolysis enzymes in thymocytes in response to various apoptotic
stimuli.
39
those glycolysis enzymes could be degraded after cleavage by caspase-l in the above two
conditions. lndeed, caspase substrates are reported to be degraded after cleavage
(Demontis et al., 2006; Ditzel et al., 2003; Du et al., 2005). Caspases initiate degradation
of their substrates and target them to be recognized by the ubiquitinlproteosome system.
Accumulating evidences link the ubiquitinlproteosome pathway to caspase function and
many researchers have implied a function of caspase-3 in the degradation of proteins
under pathogenic conditions (Mitch, 2007; Ottenheijm et al., 2006). The involvement of
caspase-l in the degradation events has not been previously reported.
In vivo cleavage of certain substrates by caspases is cell type specific and
stimulus-specific. For example, GATA-l is cleaved by caspases in erythroid cells
undergoing apoptosis in response to erythropeitin (EPO) deprivation or death receptor
stimulation ((De Maria et al., 1999), but remains uncleaved in erythroid cells undergoing
terminal differentiation (Zermati et al., 2001); actin can be cleaved by caspases in U397
leukemia cells, neurons and thymocytes, but not in other cell types undergoing apoptosis
(Eamshaw et al., 1999). So, this raises the difficulty of capturing the in vivo cleavage
event of a known in vitro caspase substrate.
In addition to the above-described caspase-l activation systems, few other caspase-l
activation systems remain to be tested and in which we could examine the cleavage of the
glycolysis enzymes. Caspase-l knockout mice develop normally and appear healthy and
fertile, but thymocytes from caspase-l deficient mice are more resistant to apoptosis
induced by anti-CD95 antibody (Kuida et al., 1995; Li et al., 1995). This suggests a
non-redundant function of caspase-l in this cell type. It would be worthwhile to explore
the cleavage of the glycolysis enzymes in thymocytes in response to various apoptotic
stimuli.
39
Although the role of caspase-1 in development is minor, its role in neuronal pathological
cell death appears to be significant and non-redundant. Dorsal root ganglion neurons in
caspase-1 deficient mice are partially resistant to apoptosis induced by nerve growth
factor deprivation (Friedlander et al., 1997b). Excessive apoptosis plays an important role
in neurodegenerative diseases as well as following cerebral ischemia and head trauma
(Friedlander et al., 1997a; Friedlander et al., 1997b; Hara et al., 1997). There is ample
evidence that suggests that caspase-1 plays a unique role in neuronal apoptosis
(Friedlander et al., 1997b). As well, it has been also proposed that caspase-1 acts as an
apical mediator of apoptosis during hypoxia-induced neuronal cell death (Zhang et al.,
2003a).
Hypoxia is a condition of low level of oxygen in tissues. A major intracellular adaptation
to severe hypoxia is the transition from oxidative phosphorylation to glycolysis as the
principal means of generating ATP. Under this condition, the hypoxia-inducible factor
(HIF) pathway is switched on. In order to obtain enough ATP, cells increase their rate of
glycolysis through stabilization of HIF, which induces the expression of the glucose
transporter GLUT1 as well as that of certain glycolysis enzymes (Semenza et al., 1994).
All of the five glycoysis enzymes that are targeted by caspase-1 are known to be HIF-1
transcriptional targets during hypoxia (Graven et al., 1999; Semenza et al., 1996). The
role of glycolysis enzymes becomes essential for cell survival in this condition. It is
therefore important to investigate the function of caspase-1 on these glycolysis enzymes
in this context.
Tumor cells display a high rate of glucose uptake and glycolysis even under normal
oxygen pressure (Warburg, 1930). Accelerated glycolysis ensures ATP levels compatible
with demands of fast proliferating tumor cells in hypoxic environment. This metabolic
strategy is commonly observed across tumors (Gatenby and Gillies, 2004). The increased
40
Although the role of caspase-l in development is minor, its role in neuronal pathological
cell death appears to be significant and non-redundant. Dorsal root ganglion neurons in
caspase-l deficient mice are partiaIly resistant to apoptosis induced by nerve growth
factor deprivation (Friedlander et al., 1997b). Excessive apoptosis plays an important role
in neurodegenerative diseases as weIl as foIlowing cerebral ischemia and head trauma
(Friedlander et al., 1997a; Friedlander et al., 1997b; Rara et al., 1997). There is ample
evidence that suggests that caspase-l plays a unique role in neuronal apoptosis
(Friedlander et al., 1997b). As weIl, it has been also proposed that caspase-l acts as an
apical mediator of apoptosis during hypoxia-induced neuronal ceIl death (Zhang et al.,
2003a).
Rypoxia is a condition of low level of oxygen in tissues. A major intraceIlular adaptation
to severe hypoxia is the transition from oxidative phosphorylation to glycolysis as the
principal means of generating ATP. Under this condition, the hypoxia-inducible factor
(RIF) pathway is switched on. In order to obtain enough ATP, ceIls increase their rate of
glycolysis through stabilization of RIF, which induces the expression of the glucose
transporter GLUTI as weIl as that of certain glycolysis enzymes (Semenza et al., 1994).
AlI of the five glycoysis enzymes that are targeted by caspase-l are known to be RIF-I
transcriptional targets during hypoxia (Graven et al., 1999; Semenza et al., 1996). The
role of glycolysis enzymes becomes essential for cell survival in this condition. It is
therefore important to investigate the function of caspase-l on these glycolysis enzymes
in this context.
Tumor cells display a high rate of glucose uptake and glycolysis even under normal
oxygen pressure (Warburg, 1930). Accelerated glycolysis ensures ATP levels compatible
with demands of fast proliferating tumor cells in hypoxic environment. This metabolic
strategy is commonly observed across tumors (Gatenby and Gillies, 2004). The increased
40
glucose uptake could play a role in protection from apoptosis by rendering tumor cells
independent of growth factor (Vander Heiden et al., 2001).Inhibition of glucose
metabolism enhances death receptor mediated apoptosis in tumor cells (Munoz-Pinedo et
al., 2003). One strategy for tumor cell propagation is through repression of caspases
(Kurokawa et al., 1999; Teitz et al , 2001). Since we have shown that caspase-1 cleaves
glycolysis enzymes in vitro, we would ask a question: Do tumor cells block caspase-1
activation to maintain their glycolytic advantage? To address this hypothesis, we can try
to induce activation of caspase-1 in tumor cells and examine the cleavage of glycolysis
enzymes and the relevance of these cleavage events to apoptosis. Previous researchers
have shown that caspase-1 dependent apoptosis is induced by doxorubicin treatment of
MCF-7 cells (a breast carcinoma cell line) (Thalappilly et al , 2006). We can use this
system to test our hypothesis.
41
glucose uptake could play a role in protection from apoptosis by rendering tumor cells
independent of growth factor (Vander Heiden et al., 2001).Inhibition of glucose
metabolism enhances death receptor mediated apoptosis in tumor cells (Munoz-Pinedo et
al., 2003). One strategy for tumor cell propagation is through repression of caspases
(Kurokawa et al., 1999; Teitz et al., 2001). Since we have shown that caspase-1 cleaves
glycolysis enzymes in vitro, we would ask a question: Do tumor cells block caspase-1
activation to maintain their glycolytic advantage? To address this hypothesis, we can try
to induce activation of caspase-1 in tumor cells and examine the cleavage of glycolysis
enzymes and the relevance of these c1eavage events to apoptosis. Previous researchers
have shown that caspase-1 dependent apoptosis is induced by doxorubicin treatment of
MCF -7 cells (a breast carcinoma cell line) (Thalappilly et al., 2006). We can use this
system to test our hypothesis.
41
Figure 4
1s t dimension gel 2"d dimension gel T a r g e t P r o t e l n s d r oP under diagonal line
Figure 4 Schematic of Diagonal Gel Approach
Whole cell lysates are separated in 1st dimension SDS-PAGE, gel lanes are excised and
dehydrated, and then re-hydrated in presence of protease of interest, allowing in-gel
cleaving of substrates by protease. The processed gel lanes are further resolved by 2nd
dimension SDS-PAGE. The majority of proteins migrate along diagonal line, whereas
fragments of substrates cleaved by protease drop under the diagonal line. The dropped
proteins are identified by MALDI-TOF.
42
Figure 4
Gel incubated Target with caspase-1 prote in
~ --..
1st dimension gel 2nd dimension gel
Figure 4 Schematic of Diagonal Gel Approach
Diagonal line
Target proteins drop
under diagonal line
Whole celllysates are separated in lst dimension SDS-PAGE, gellanes are excised and
dehydrated, and then re-hydrated in presence of protease of interest, allowing in-gel
c1eaving of substrates by protease. The processed gel lanes are further resolved by 2nd
dimension SDS-PAGE. The majority of proteins migrate along diagonal line, whereas
fragments of substrates c1eaved by protease drop under the diagonal Hne. The dropped
proteins are identified by MALDI-TOF.
42
Figure 5
No caspase
B Caspase-3
Figure 5 Diagonal Gel with Caspase-3
Whole cell extracts from PBMC were loaded on gel and ran in 1st dimension
SDS-PAGE. The gel lane was incubated with or without caspase-3 for overnight,
then ran in 2nd dimension SDS-PAGE. The gels were stained with Sypro Ruby
and viewed under UV light. (A) without caspase (B) with caspase-3
43
.. ~
Figure 5
A No caspase
B Caspase-3
Figure 5 Diagonal Gel with Caspase-3
Whole cell extracts from PBMC were loaded on gel and ran in 1 st dimension
SDS-P AGE. The gellane was incubated with or without caspase-3 for overnight,
then ran in 2nd dimension SDS-PAGE. The gels were stained with Sypro Ruby
and viewed under UV light. (A) without caspase (B) with caspase-3
43
Figure 6
Figure 6 Caspase-1 Substrate IL-1 P Revealed by Western Blot in Diagonal Gel
THP-1 cells were pretreated with LPS for overnight, 400 [ig proteins from whole cell
lysates were resolved in 4-12% SDS-PAGE, the lane was hydrated and then incubated
with human recombinant Caspase-1 (25 [xg) overnight, and again resolved with 4-12%
SDS-PAGE gel, western blot using IL-1 0 antibody. The dash line represents diagonal.
The dot drew on diagonal line represents the proform of IL-1 0 (34 KDa).
44
(~.
Figure 6
150-.r-______________________________________ ~
100 75
50
37
25
20
15
10 ~-----------------------------------------
Figure 6 Caspase-l Substrate IL-l ~ Revealed by Western Blot in Diagonal Gel
THP-1 cells were pretreated with LPS for overnight, 400 !-tg pro teins from whole cell
lysates were resolved in 4-12% SDS-PAGE, the lane was hydrated and then incubated
with human recombinant Caspase-1 (2S!-tg) overnight, and again resolved with 4-12%
SDS-PAGE gel, western blot using IL-1 13 antibody. The dash Hne represents diagonal.
The dot drew on diagonal li ne represents the proform ofIL-1 13 (34 KDa).
44
Figure 7
w ®
w
HSP-90
HSP-90 Lactoferrin
HSP-70
MCMS Transketolase
HSP90
HSP-60 Cataiase Pyruvate kinase Thyroid hormone BP AnnexinAil ,£3,
HSP-60 Cataiase Pyruvate kina
ImRNP-G MHCclassIHLACw0803
Actiu Elongation factorTu
Transaldolase i
Ail in ® MHC class I cw2
97,4
Chain D myeloperoxidase j Dihydrolipoamide dehydrogenase
Chain D myeloperoxidase | Itfitoeh. serine hydroxymethyltransferas^
Leucine aminopeptidase
>\ j 1 • \ i ti
i'vnu it< Mm i. Vll'^nlhas.. H lr<tns|K>it i I 7
Alpha hemoglobin
®
OAPDH @ hnRNPA2,hnRNPAl. Cappissj: pririem sir>ta
GAPDH @
Migration-inducing gene 10
GAPDH hnRNPA2 ANXA2 PPPIA
©
Horf-6 Stomatin
18
Figure 7 Map of Caspase-3 Digested Diagonal Gel
Whole cell extracts from PBMC were subjected to SDS PAGE gel and then digested with recombinant caspase-3. 2nd dimension gel was stained with silver. The dots marked were analyzed by MALDI-TOF.
Figure 7
HSP-60 Cataia."\:" Pyruvate kinasè
hnRNP,G MHC c1ass 1 HLA Cw0803
97.4
... (41 Chain D myeloperoxidase
Dihydrolipoamide dehydrogenase
TrdHsahtobsl..~ ~
igration-inducing gene 10
GAP!)H hnRt\P Al ANXA2 PPPIA
Horf-6 Stomatin
Figure 7 Map of Caspase-3 Digested Diagonal Gel
Whole cell extracts from PBMC were subjected to SDS PAGE gel and then digested with recombinant caspase-3. 2nd dimension gel was stained with silver. The dots marked were analyzed by MALDI-TOF.
45
Figure 8
A
B
Caspase - l
Channels, t ransporter
and Signaling 20%
Glycolysis &\ Energy \
metabolism \ 23% \
Ca
Channels,
transporter and Signaling
16% ^
Gycolysis & \ Energy \
metabolism \ 21% \
spa se -3
others
5%
Immune proteins 7%
s u b s t r a t e s
Cytoskeletal and Membrane
proteins
H H ^ ^ Chaperons and H | ^ | | 8 E ^ \ . Proteins for
^ ^ ^ R | | I : M \ folding ^^W fllSfS. 9% ^^^^^^p *?$^S2~^;\
^ • ^ /Transcription ^ ^ ^ k / and Nuclear
^ ^ ^ ^ ^ / proteins ^ ^ ^ ^ k / 19%
Translation Machinery
18%
s u b s t r a t e s
Cytoskeletal and Membrane
- _ _ „ ^ proteins
Unfe^. 21%
L^^^H^^HRPiA Chaperons and l ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ p ^ j Proteins for
^ - S B S ^ T T w ^ l folding
A ^""/ 7%
^ ^ ^ L ; / Transcription ^ ^ ^ ^ - - ^ and Nuclear "Translation proteins
Machinery igo/0
7%
Figure 8 Pie Chart of Caspase-l, -3 Substrates
(A) Caspase-l substrates (B) Caspase-3 substrates
46
(\ Figure 8
A
B
Caspase-l substrates
Channe1s, transporter
and Signa1ing 20%
Energy metabo1ism
23%
Cytoskeleta1 and Membrane
Translation Machinery
18%
Chaperons and Proteins for
fol ding 9%
proteins 19%
Ca spase-3 substrates
Channels,
transporter and
Signaling 16%
Glycolysis &
Energy
metabolism
21%
7%
others
5%
Machinery
7%
Cytoskeletal and
Membrane
proteins
21%
Chaperons and Proteins for
folding 7%
proteins
16%
Figure 8 Pie Chart ofCaspase-l, -3 Substrates
(A) Caspase-l substrates (B) Caspase-3 substrates
46
Figure 9
Caspase-1 Caspase-3
Figure 9 Venn Diagram Comparing Caspase-1 and Caspase-3 Substrates
(A) Overlapping of caspase-1, caspase-3 substrate in total proteins identified by diagonal gel approach
(B) Overlapping of caspase-1, caspase-3 substrates in cytoskeletal proteins identified by diagonal gel approach
47
Figure 9
Caspase-l Caspase-3
A
58
B
Figure 9 Venn Diagram Comparing Caspase-l and Caspase-3 Substrates
(A) Overlapping of caspase-l, caspase-3 substrate in total proteins identified by diagonal gel approach
(B) Overlapping of caspase-l, caspase-3 substrates in cytoskeletal proteins identified by diagonal gel approach
47
Figure 10
Glucose ATP.— .
*.*/ fllucoklnsse
f Glucose-6-phosphate
TpltosphohiKBse 1 SORltSi'iSP
Fructose-6-phosphate
^ w pbospho-
Fructose-1,6-bisphosphate
ATP-
ftOP«
seraldehyde-3-p
^Ntalycei
hydroxyacetone
ceraM eh yd e-3-p h o sph ate dehydrogenase
1,3-bisphosphogly cerate
} | phosphoglycerate ^ p ^ ) - * — ^ 1 k""ase
3 phosphoglycerate Jphosphoglyceiate * mutase
2-ph ospho gly cerate
Ljen
pyruvate
Pyruvate I | lactate I dehydrogenase
Lactate
Figure 10 Caspase-1 Targeted Substrates in Glycolytic Pathway
Glycolytic pathway converts glucose to pyruvate via glycolysis enzymes. Under anaerobic condition, pyruvate is converted to lactate to re-generate NAD+- In this pathway, 6 out of total 11 glycolytic enzymes are among our list of casapase-1 substrates, which are marked with red square.
Figure 10
NADH
Glucose ATP~~heXOldn1lSe
glucokinllse AOP
GI ucose-6-phosphate ~Ph?$PlmheXO$O • lSomClll$o
F~~~OSe-6'PhosPhate phospho-
AOP fructokltlllSa-'
Fructose~hOSPhaœ
~-~. ... h h- 1 fi&-! Olhydroxyaçetone e-.>-p osp ... e phosphate
1 !;:st'SPhO.S hoglycerate
phosplwgtycerate @ kinase
3-PhOSPlo9,vcorate phosphogllfcerme
mmase 2-phosphoglycerate
;lenOlase 1 Phospho nolpyruvate ADP
Pyruvate
!~ Lactate
Figure 10 Caspase-l Targeted Substrates in Glycolytic Pathway
Glycolytic pathway converts glucose to pyruvate via glycolysis enzymes. Under anaerobic condition, pyruvate is converted to lactate to re-generate NAD+· In this pathway, 6 out of total Il glycolytic enzymes are among our list of casapase-1 substrates, which are marked with red square,
48
/"' Figure 11
Casp-1
7 5 -
5 0 -
3 7 -
25 _
20 ~
15 -
' • . ' ' . ' • '
; * 100
75
50
37
25 20
IL-1j8 Aldolase a - Enolase
Casp-1
ioo-75 _
50 _
37 "
25 -
20 -
«^jg|i|jj$|#~ -•wMq»vw»-
•o^pp -mmm* < -
ioo-7 5 -
5 0 _
3 7 "
2 5 -
2 0 "
i
«fe40fe
TIM GAPDH
49
Figure 11
Casp-l
75
37
Casp-l -
100
75
50
37
+
IL-1/3
+
TIM
100 -
75
50
37
25 20
100
75
25
20
+ +
100
75
50
Aldolase (1- Enolase
+
GAPDH
49
Figure 11 Western Blot of in vitro Cleavage of Glycolysis Enzymes by caspase-1
Whole cell extracts from THP-1 were incubated with or without recombinant caspase-1
for 3 hours at 37°C and resolved in 4 - 12% SDS-PAGE, western blot uses antibodies
against mature IL-113 , aldolase, a - enolase, TIM and GAPDH. Open arrows indicate
full-length proteins, and closed arrows indicate the cleavage fragments.
F
50
Figure 11 Western Blot of in vitro Cleavage of Glycolysis Enzymes by caspase-l
Whole cell extracts from THP-l were incubated with or without recombinant caspase-l
for 3 hours at 3TC and resolved in 4 - 12% SDS-PAGE, western blot uses antibodies
against mature IL-l 13 ,aldolase, a - enolase, TIM and GAPDH. Open arrows indicate
full-length proteins, and closed arrows indicate the cleavage fragments.
F
50
Figure 12
A
1 0 0 -75 -50 -
37 -
25 _
20 _
15 -
10 -
/
• > • • » . . & .
^™^^^£V "̂ K^™
<
4"
" ' . . * • - ; -
a-
_aspase-l
# #
*
. ' i . i : "KR.-V. '
Enolase
4"
... . ; y.~-•
-9
!.:V'f-
. .
< -
<-
B /
£ Caspase-1
4* /
4" /
100 -75 -50 -
37 -
25 _
20 _
15 -
10 -
^r&s^s*' ' ' ^ISHI
; .">:- i&J&yiiQ
.,,
Aldoase
r^.
51
r-"
Figure 12
A
B
100· 75 50 37
25
20
15
10
100 75 50 37
25
20
15
10
~ "'" J!
vS §
~ "'" J!
vS §
~ Caspase-l $ 0
~ ~ ~ ~ ~ ~ ~ ~ ~ l"'5 00 '"
Caspase-l ~
$-~ ~ 0 ~
~ ~ ~ ~ l"'5 00
51
Figure 12 Western blot of Comparing Cleavage of Glycolysis Enzymes by Caspase-1 and -3
Whole cell extracts from THP-1 cells were incubated with or without caspase-1/ -3 of
indicated amount for 3 hours at 37 °C, and resolved in 10% SDS-PAGE, cleavgage of
a - Enolase and aldolase was assessed by western blot. Open arrows indicate full-length
proteins, and closed arrows indicate the cleavage fragments.
(A) anti- a -Enolase
(B) anti- aldolase
52
Figure 12 Western blot of Comparing Cleavage of Glycolysis Enzymes by Caspase-l and -3
Whole cell extracts from THP-l cells were incubated with or without caspase-ll -3 of
indicated amount for 3 hours at 37 oC, and resolved in 10% SDS-PAGE, c1eavgage of
a - Enolase and aldolase was assessed by western blot. Open arrows indicate full-length
proteins, and c10sed arrows indicate the c1eavage fragments.
(A) anti- a -Enolase
(B) anti- aldolase
52
Figure 13
150.
100.
75.
50-
37.
25-
20.
150 •
100-75 .
#
t
50 " j j j | •/.J&,,
i
'<!§'
Internal translation
I <3 i 100-75 .
#
m I m
<f
ISllslHllSllSlsl
50
37
15 J
Aldolase Pyruvate kinase
53
("
Figure 13
37
25
20
15
150
37
25
TlM
Aldolase
GAPDH
-. "'1
100 75 .
50 .
37 ,
25 20 ;
15 ;
Pyruvate kinase
53
Figure 13 Comparison of Cleavage of ITT Glycolysis Enzymes by Caspase-1 and -3
in vitro transribed and translated 35S labeled PRAP, GAPDH, TIM, a - Enolase,
aldolase and pyruvate kinase were incubated with or without recombinant caspase-1
(170ng) or caspase-3 (290ng) for 4 hours at 37 °C, resolved in 4 - 12% SDS PAGE
(TIM was resolved in 12% SDS PAGE ) and viewed by autoradiography. Opened arrows
indicate full-length proteins, closed arrows indicate cleaved fragments.
54
•
Figure 13 Comparison of Cleavage of ITT Glycolysis Enzymes by Caspase-l and -3
in vitro transribed and translated 35S labeled PRAP, GAPDH, TIM, a - Enolase,
aldolase and pyruvate kinase were incubated with or without recombinant caspase-1
(l70ng) or caspase-3 (290ng) for 4 hours at 37 oC, resolved in 4 - 12% SDS PAGE
(TIM was resolved in 12% SDS PAGE) and viewed by autoradiography. Opened arrows
indicate full-length proteins, closed arrows indicate cleaved fragments .
54
Figure 14
Caspase-I(ng) *
Internal translation
a - Enolase
B
Caspase-I(ng)
150 -
100 -
75 -
50 ~
3 7 -
s>
•m
^
MI
<?
•m
#
m
^ ^ <b*
25 -
20 -
^m '• '• .>
".- ^
T f •"*"
• , '•'
r<
i f IBP i f l i p p i l ' '"PHI P I iii!!i!iii!iijijiijiii;'nw"
. ™;,'$ &*<»* *' ̂ 4> k,-* ^*^«*# *--• *'-
•? - l . * ' > - ^-Possible internal translation
Aldolase
55
Figure 14
A
Caspase-1 (ng)
B
150 100
75
50
37
25 20
~ ~ ~ ~~ ~~
'"
,~' ;~
0: - Enolase
Caspase-1(ng) ~ ~ ~ ~~
150, 100.
75
50 '
37 '
25 .
20 .
Aldolase
~~
" i' n;
InternaI translation
' .•.. :: Je internai ~ !i§i'!i!slation
55
Figure 14
Caspase-1 (ng) * ^ ^ <? •? ^ <b* ^ 150—
100
75 ""
50 —
37 ""
25 — 20 ""
15 —
; ;;:;::m
< •
- | Cleavage -J fragments
D
GAPDH
Caspase-1 (ng) N n, <J s> fc* ^* ^ N <3 »$> <$>
Pro- IL-1 |
56
r Figure 14
C ~ç::, ~ç::, ~ ç::,ç::, Caspase-1 (ng) ç::, ~ ~ ~ç::, '" " ftj '\
150-
100
75 -
50 -
37 -
25 -20 - ] Cleavage
15 -fragments
GAPDH
D
Caspase-1 (ng) " ç::,~ " ~ ~ ç::, ç::,. ç::,. " " 1
56
Figure 14 Calibrating cleavage of ITT Glycolysis Enzymes by Caspase-1
in vitro transribed and translated 35S labeled a - Enolase, aldolase, GAPDH, TIM and pyruvate
kinase were incubated with or without recombinant caspase-1 of indicated amount for 4 hours at 37
°C , resolved in 4 - 12% SDS PAGE and viewed by autoradiography. Opened arrows indicate
full-length proteins, closed arrows indicate cleaved fragments.
(A) a - Enolase ITT
(B) Aldolase ITT
(C) GAPDH ITT
(D) ProIL-lP ITT
57
Figure 14 Calibrating cleavage ofITT Glycolysis Enzymes by Caspase-l
in vitro transribed and translated 35S labeled a - Enolase, aldolase, GAPOH, TIM and pyruvate
kinase were incubated with or without recombinant caspase-l of indicated amount for 4 hours at 37
oC , resolved in 4 - 12% SOS PAGE and viewed by autoradiography. Opened arrows indicate
full-Iength proteins, closed arrows indicate cleaved fragments.
(A) a - Enolase ITT
(B) Aldolase ITT
(C) GAPOH ITT
(0) Pro IL-l fi ITT
57
Figure 15
A
B
VIHD 166 KTVD 189
EKYiD141
20 KDa • !
30KBS
rr KWED198
20 KDa
GAPDH
189
Human NFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQN Mouse NFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGAAQN Cow HFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGAAQN yeast AFGIEEGLMTTVHSMTATQKTVDGPSHKDWRGGRTASGN
Caspase-1
7 5 "
5 0 -
3 7 "
25 — 20 —I
wt D189A
•521 GAPDH
58
Figure 15
A
VIHD166 KTVD189
-20KDa -20KDa
B 189
c
Human NFGIVEGLMTTVHAITATQKTV'tPSGKLWRDGRGALQN
Mouse NFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGAAQN
Cow HFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGAAQN
yeast AFGIEEGLMTTVHSMTATQKTVDGPSHKDWRGGRTASGN
Caspase-1
50
37
25 20
wt D189A
+ +
GAPDH
58
Figure 15 Identification of Caspase-1 Cleavage Site of in GAPDH
(A) Analysis of potential cleavage sites for GAPDH
(B) Alignment of region containing caspase-1 cleavage site in GAPDH from different
species
© Blockage of cleavage of GAPDH by caspase-1 in GAPDH D189A mutant
In vitro transcribed and translated 35s labeled GAPDH and GAPDH D189A were
incubated with or without 170 ng recombinant caspase-1 for 4 hours at 37 °C ,
resolved in 4 - 12% SDS PAGE and viewed by autoradiography. Opened arrows
indicate full-length proteins, closed arrows indicate cleaved fragments.
59
Figure 15 Identification of Caspase-1 Cleavage Site of in GAPDH
(A) Analysis of potential cleavage sites for GAPDH
(B) Alignment of region containing caspase-l cleavage site in GAPDH from different
speCIeS
© Blockage ofcleavage ofGAPDH by caspase-l in GAPDH D189 A mutant
In vitro transcribed and translated 35s labeled GAPDH and GAPDH D189A were
incubated with or without 170 ng recombinant caspase-l for 4 hours at 37°C ,
resolved in 4 - 12% SDS PAGE and viewed by autoradiography. Opened arrows
indicate full-length proteins, closed arrows indicate cleaved fragments.
59
Figure 16
HIAD13*
a -Enolase
B
D136A D238A D266A D286A
Internal translation
<* -Enolase
60
Figure 16
A
-20KDa
B
D136A Caspase-l . +
150 100 75 .
50
37
25 20
15
47.4KDa
Cl! - Enolase
D238A D266A
+- d-e
Π~Enolase
-20KDa
D286A
Internai translation
60
Figure 16 Identification of Caspase-1 Cleavage Site in a - Enolase
(A)Analyzing cleavage site for a - Enolase
(B)Failure to block cleavage by caspase-1 in 4 potential cleavage site mutants of
a-Enolase
In vitro transcribed and translated 35s labeled a - Enolase and its mutants were incubated
with or without 170 ng recombinant caspase-1 for 4 hours at 37 °C , resolved in 4 - 12%
SDS PAGE and viewed by autoradiography. Opened arrows indicate full-length proteins,
closed arrows indicate cleaved fragments.
61
Figure 16 Identification of Caspase-l Cleavage Site in a - Enolase
(A)Analyzing c1eavage site for a - Enolase
(B)Failure to block c1eavage by caspase-1 in 4 potential c1eavage site mutants of
a-Enolase
In vitro transcribed and translated 35s labeled a - Enolase and its mutants were incubated
with or without 170 ng recombinant caspase-1 for 4 hours at 37 oC ,resolved in 4 - 12%
SDS PAGE and viewed by autoradiography. Opened arrows indicate full-Iength proteins,
c10sed arrows indicate c1eaved fragments.
61
/•"""V
Figure 17 #
* 4> v
75
50
37
25 20
Caspase-1
„<? *
y • ^ ^
aldolase
62
Figure 17
75
50
37
25 20
100 75 50
37
25 20 15 10
100 75 50
37
25 20
Caspase-l
J' D ~
4;;)~
a-Enolase
-
aldolase
62
Figure 17 Western blot of Glycolysis Enzymese in ATP/Nigericin treated THP-1 Cells
THP-1 cells were pre-treated with PMA (20ng/ml) overnight and then treated with LPS (lug/ml) for 3
hrs and further treated with ATP or nigericin to activate caspase-1. Cells were lysed in SDS loading
buffer and resolved in 4-12 % SDS PAGE. Western blot was performed using antibodies against
caspase-1, a - enolase and aldolase.
63
Figure 17 Western blot of Glycolysis Enzymese in ATPlNigericin treated THP-l CeUs
THP-l cells were pre-treated with PMA (20ng/ml) overnight and then treated with LPS (l~g/ml) for 3
hrs and further treated with ATP or nigericin to activate caspase-l. Cells were lysed in SDS loading
buffer and resolved in 4-12 % SDS PAGE. Western blot was performed using antibodies against
caspase-l, a - enolase and aldolase.
63
/to
-i
| to
A-, *. 'tt
HO
SS
t •
i
li .11
if
j
a> « o
c U
l
II I
I—I
I II
Oin
o
r> ®
t~ «r, *n
in
o
ino
vi o
vi o
)
Figure 18
A THP-1 PMA THP-1 PMA-LPS THP-1 PMA-LPS-Salm 1:1 THP-1 PMA-LPS-Salm 1:5 THP-1 PMA-LPS-Salm 1:10 10
80
Ji=60 u rn rn
40'
20
1<f1 1à
B ~ ,q;
50
37
25 20
15 10
1ei' W1 1rf 1<1' FL1-H
Lt" Salmonella ",' "' ~ ~ ~ ~ ~ =,,'0
~ ..... ",' ",. ~ #' :t ;s. :t l-~ q q $~'"
IL-1~
::ç u (1) rn
01 i ; i ; i ; 1 0 1 ; i ; i Oh 1 1d 1I:i' 1a 1a 1& 1~ 1a 1a 1d 18 1d 1a 1d 1d
FL1-H FL 1-H FL1-H
7S -
~" ~
#' ~~ ~ ~"
50-1 __
37
25 -
20 -
Salmonella ~
~ ... ~ :-. ", =-- ..::;: ",''';.. ",' .
~ :t ~ ~~ q q q ~
Caspase-1
100 75 50
37
25 20 15 10
~ ,," Salmonella ~ .' n f$' ",. " ,. _.;$' .... ~ '", .~ :..
... 'It -$
~,,~ Il 1
Aldolse
~.
~ ~
100 75 50
37
25 20
15 10
100'
o 1 ; i ; i 1
1d' 101 1a la la' FL1-H
~ Salmonella ~ .... 1" ~ ... ~ • ~ ,. ,.' ,"f ~'"
~ J i;- ~ Q'" ~. ~~~ q$$~ .. (,;
--------
a-Enolase
)
64
)
Figure 18 Depletion of Glycolysis Enzymes in Salmonella infected THP-1 cells
THP-1 cells were pre-treated with PMA and primed with LPS and then infected with Salmonella. Activation of caspase-1 was
monitored by FACS analysis with FLICA08*"1. Cells were lysed in SDS-PAGE buffer and lysates resolved on a 4-12% SDS PAGE.
Western blot was performed using antibodies against caspase-1, mature IL- 16, oc-enolase, and aldolase.
(A) FACS with FLICAcasp-1
(B) Western Blot with antibodies against caspase-1, mature IL-16, a-enolase and aldolase.
) )
Figure 18 Depletion of Glycolysis Enzymes in Salmonella infecfed THP-1 cells
THP-1 cells were pre-treated with PMA and primed with LPS and then infected with Salmonella. Activation of caspase-1 was
monitored by FACS analysis with FLICAcasp-l. Cells were lysed in SDS-PAGE buffer and lysates resolved on a 4-12% SDS PAGE.
Western blot was performed using antibodies against caspase-1, mature IL- 1~, a-enolase, and aldolase.
(A) FACS with FLICAcasp-1
(B) Western Blot with antibodies againstcaspase-1, mature IL- 1~, a-enolase and aldolase.
65
Figure 19
** -^
Casp-1
LPS
00 -75 -
50 "
37 -
25 -20 -15 "
•
.
IL-1B
100_
75 Z
50 -
37 "
25 -20 -15 -
^
«?
-?
«r
**
*«»
a - Enolase
'-> -
50-
37 "
25 -20"
15 -10"
• -• ^mm*
!
a - actin
GAPDH TIM
66
~. Figure 19
~ ~ ~ ~ ~ ~
LPS ~ ~ ~ ~ ~ ~ ~ ~ ~
75 75 _ 75
50 50 - 50 _ 1j$:. ,. • .-. 37 - 37 -
,,",' .. ',)"
37 i~"
25 - 25 - - '. .-25 20 -.,... - 20 -20
15 : 15 15 - 10 10 10-
Casp-l IL-ID a - actin ~ ~
LPS f'V ~ 75 _ 100 -75 _ 50 ... _"',,1'
50 -37 ... -37 -
25 -15 20 -10- 15 -
Aldolase a - Enolase ~ ~ ~ ~
LPS ~ ~ ;: ~ :: ~ 75 _
100 _ " .,
75 - 50 -
50 - 37 -
37 - + 25 -20 - .. . ~ -25 -
20 - 15 -15 - 10-
GAPDH TIM
66
Figure 19 Depletion of glycolysis Enzymes in Mice Muscle with Septic shock
Mice were injected intraperitoneally with 20mg/kg LPS to induce septic shock. The mice
were killed 12 or 24 hrs post-injection. Diaphragm muscle was excised, and proteins
were extracted and subjected in 4-12 SDS PAGE, western blot were performed using
antibodies against caspase-1, IL-1B, a-enolase, aldolase, GAPDH and actin.
67
Figure 19 Depletion of glycolysis Enzymes in Mice Muscle with Septic shock
Mice were injected intraperitoneally with 20mglkg LPS to induce septic shock. The mice
were killed 12 or 24 hrs post-injection. Diaphragm muscle was excised, and proteins
were extracted and subjected in 4-12 SDS PAGE, western blot were performed using
antibodies against caspase-l, IL-lB, a-enolase, aldolase, GAPDH and actin.
67
Figure 20
Fold
4
3.5
3
2.5
2
1.5
1
0.5
0
r
-
Lactate Production
1 1 1
< , # <v* ^
Figure 20 Change of Glycolysis Rate in THP-1 Cells After Salmonella Infection
THP-1 cells were pretreated with PMA( 20 ng/ml) for 18 hrs, and infected with
Salmonella (moi 1:10) for 4 hrs in the presence or absence of YVAD-FMK. Rate of
glycolysis was assessed by Lactate production
68
Figure 20
Lactate Production
4
3. 5 3
2.5 ~ 2 ~
1.5
1
O. 5 0 1 1
Figure 20 Change of Glycolysis Rate in THP-l CeUs After Salmonella Infection
THP-1 cells were pretreated with PMA( 20 ng/ml) for 18 hrs, and infected with
Salmonella (moi 1:10) for 4 hrs in the presence or absence of YVAD-FMK. Rate of
glycolysis was assessed by Lactate production
68
Table 1 Caspase-1 and -3 substrates obtained by diagonal gel approach
Protein
Cytoskeletal and membrane proteins
adenylyl cyclase-associated protein
actin-related protein 3
cellular myosin heavy chain
coronin-like protein
cytokeratin 9
F-actin capping protein a-1 subunit
filamin 1
gelsolin isoform a
gelsolin isoform b
keratin 1
stomatin peptide
tropomyosin isoform
vimentin
accession
number
gi|5453595
gi|5031573
gi|553596
gi| 1002923
gi|435476
gi|5453597
gi|4503745
gi|4504165
gi|38044288
gi|7331218
gi|181184
gi|9508585
gi|37852
Function
membrane protein
membrane protein
cytoskeletal protein
cytoskeletal protein
assembly/signaling
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
membrane protein
cytoskeletal protein
cytoskeletal protein
Casp-1
X
X
X
X
X
X
Casp-3
X
X
X
X
X
X
X
X
X
Known
substrate
X
X
X
X
X
X
X
--)
~)
/
Table 1 Caspase-l and -3 substrates obtained by diagonal gel approach
Protein accession Function Casp-l Casp-3 Known
number substrate
Cytoskeletal and membrane pro teins
adenylyl cyclase-associated protein gij5453595 membrane protein X X
actin-related protein 3 gij5031573 membrane protein X
cellular myosin heavy chain gij553596 cytoskeleta1 protein X X
coronin-like protein gij1002923 cytoskeleta1 protein X
assemb1y/signa1ing
cytokeratin 9 gij435476 cytoske1eta1 protein X
F-actin capping protein a-1 subunit gij5453597 cytoskeleta1 protein X X X
filamin 1 gij4503745 cytoske1eta1 protein X X
ge1solin isoform a gij4504165 cytoskeleta1 protein X X
gelsolin isoform b gij38044288 cytoskeleta1 protein X X
keratin 1 gij7331218 cytoske1etal protein X X
stomatin peptide gij181184 membrane protein X
tropomyosin isoform gij9508585 cytoske1eta1 protein X
vimentin gij37852 cytoskeleta1 protein X X
69
Protein
ot-actin
a-tubulin
P-tubulin
y-actin
Chaperons and proteins for folding
Calnexin
calreticulin precursor
glutathione transferase
gp96 precursor
accession number
gi| 178027
gi| 15010550
gi| 1297274
gi| 178045
gi| 13097684
gi|4757900
gi|87564
gi| 15010550
HSP27 gi|662841
HSP60 gi|77702086
HSP70 protein 8
gi|5729877
Function
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
cytoskeletal protein
Casp-1
X
X
X
Casp-3
X
X
X
X
Known
substrate
X
X
X
X
retains incorrectly folded
glycoproteins in ER
chaperon
protein folding
heat shock protein,
chaperone
heat shock protein,
chaperone
heat shock protein,
chaperone
heat shock protein,
chaperone
X
X
X
X
X
X
X
X
) ") /
Protein accession Function Casp-l Casp-3 Known
number substrate
a~actin gij178027 cytoskeletal protein X X
a~tubulin gij15010550 cytoske1etal protein X X X
/3~tubulin gij1297274 cytoskeletal protein X X X
y~actin gil178045 cytoskeletal protein X X X
Chaperons and proteins for folding
Calnexin gil13097684 retains incorrectly folded X X
glycoproteins in ER
calreticulin precursor gij4757900 chaperon X
glutathione transferase gil87564 protein folding X
gp96 precursor gij15010550 heat shock protein,
chaperone
HSP27 gil662841 heat shock protein, X
chaperone
HSP60 gij77702086 heat shock protein, X
chaperone
gij5729877 heat shock protein, X X X
HSP70 protein 8 chaperone
70
Protein
HSP90
protein disulfide isomerase-related protein 5
PPIB precursor
Transcription and nuclear proteins
A+U-rich element RNA binding factor
APEX nuclease
HI histone family, member 2
HI histone family, member 3
Hlb histone
accession Function
number
gi|306891 heat shock protein,
chaperone
G i| 1710248 protein folding
gi| 118090 Peptidyl-prolyl cis-trans
isomerase B precursor,
protein folding
gi|2547076 transcription
gi| 17939646 transcription
gi|4885375 structural protein of
chromatin
gij4885377 structural protein of
chromatin
gi|356168 structural protein of
chromatin
) ")
Protein accession Function Casp-l Casp-3 Known
number substrate
HSP90 gil306891 heat shock protein, X X X
chaperone
protein disulfide isomerase-related protein 5 G il1710248 protein folding X
PPIB precursor gill18090 Peptidyl-prolyl cis-trans X
isomerase B precursor,
protein folding
Transcription and nuclear proteins
A+U-rich element RNA binding factor gil2547076 transcription X
APEX nuclease gil17939646 transcription X
Hl histone family, member 2 gil4885375 structural protein of X
chromatin
Hl histone family, member 3 gi14885377 structural protein of X
chromatin
Hlb histone gil356168 structural protein of X
chromatin
71
Protein
HMG-1
accession
number
gi|968888
hnRNP 2H9B
hnRNPAl
gi|7739437
gi| 133254
hnRNP A2/B1
hnRNP C1/C2
hnRNP D
hnRNP E2
hnRNP G
hnRNP H1/H2
laminin-binding protein
pl05MCM
MCM5
gi|4504447
gi|62088634
gi|870743
gi|460773
gi|542850
gi|6065880
gi|34234
gi| 1197636
gi| 1232079
RNH gi| 12653783
Function
structural protein of
chromatin
transcription
Heterogeneous nuclear
ribonucleoprotein
Transcription
transcription
Transcription
Transcription
Transcription
Transcription
nulear protein
replication
replication
Casp-1
X
X
X
X
X
X
X
X
X
X
X
Casp
X
X
X
X
X
X
Known
substrate
X
Ribonuclease/ angiogenin X
inhibitor
72
) ') /
Protein accession Function Casp-l Casp-3 Known
number substrate
HMG-I gij968888 structural protein of X
chromatin
hnRNP2H9B gij7739437 transcription X
hnRNPAI gij133254 Heterogeneous nuc1ear X X
ribonuc1eoprotein
hnRNP A2/BI gij4504447 Transcription X X
hnRNP Cl/C2 gij62088634 transcription X
hnRNPD gij870743 Transcription X X
hnRNPE2 gij460773 Transcription X X
hnRNPG gij542850 Transcription X X
hnRNPHI/H2 gij6065880 Transcription X
laminin-binding protein gil34234 nulear protein X
plO5MCM gil1197636 replication X
MCM5 gil1232079 repli cation X X
RNH gil12653783 Ribonuclease/ angiogenin X
inhibitor
72
Protein accession
number
U2 small nuclear ribonucleoprotein polypeptide A' gi| 18605961
Translation machinery
EEFTu
EEF1A1
EEF1D
EFla-like protein
EF2
nascent-polypeptide-asso dated complex a
ribosomal protein LI3
ribosomal protein LI 8
ribosomal protein L6
ribosomal protein L7a
ribosomal protein P0
ribosomal protein S10
gi|704416
gi(48735185
gi|38522
gi| 12006049
gi|181969
gi|5031931
gi| 15431297
gi|4506607
gi|21410970
gi|4506661
gi| 12654583
gi|55665989
^
Function
transcription
Casp-1 Casp-3
X
Known
substrate
X
elongation factor Tu
elongation factor-1-a
elongation factor-1-8
elongation factor 1 a
elongation factor 2
translation
translation
translation
translation
translation
translation
translation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
73
')
Protein
U2 small nuclear ribonucleoprotein polypeptide A'
Translation machinery
EEFTu
EEFIAI
EEFID
EFla-like protein
EF2
nascent-polypeptide-associated complex a
ribosomal protein L 13
ribosomal protein L 18
ribosomal protein L6
ribosomal protein L 7a
ribosomal protein PO
ribosomal protein SI 0
accession
number
gil18605961
gil704416
gij48735185
gil38522
gil12006049
gil181969
gil5031931
gil15431297
gij4506607
gil21410970
gil4506661
gij12654583
gil55665989
Function Casp-l
transcription
elongation factor Tu X
elongation factor-l-a X
elongation factor-I-ô X
elongation factor 1 a X
elongation factor 2 X
translation X
translation X
translation X
translation X
translation X
translation X
translation X
Casp-3
x
X
X
X
X
Known
substrate
X
X
X
')
73
^ /
Protein
ribosomal protein S3
ribosomal protein S7
ribosomal protein S9
accession
number
gi|7765076
gi|337518
gi|550023
Immune proteins
CAP37 gi|240869
Coagulation factor XIIIA chain precuror
HLA-I Cw-2 a
HLA-I heavy chain
gi|119720
gi|231429
gi| 1196444
Glycolysis and Energy metabolism
2-phosphopyruvate-hydratasea-enolase gi|693933
aldolase A gi|28614
ATP synthase, ATP synthase, H+ transporting, gi|32189394
mitochondrial Fl complex, |3 subunit precursor
cationic antimicrobial
protein
blood coaguation
human leukocyte antigen
human leukocyte antigen
X
X
X
X
glucose metabolism
glycolysis
energy production
X
X
X
X
X
1
Casp-1
X
X
X
Function
translation
translation
translation
Casp-3 Known
substrate
74
)
Protein
ribosomal protein S3
ribosomal protein S7
ribosomal protein S9
Immune proteins
CAP37
Coagulation factor XIII A chain precuror
HLA-I Cw-2 a
HLA-I heavy chain
Glycolysis and Energy metabolism
2-phosphopyruvate-hydratase a-enolase
aldolase A
accession
number
gil7765076
gij337518
gil550023
gil240869
gi1119720
gil231429
gill196444
gil693933
gil28614
ATP synthase, ATP synthase, H+ transporting, gil32189394
mitochondrial FI complex, ~ subunit precursor
Function
translation
translation
translation
cationic antimicrobial
protein
blood coaguation
human 1 eukocyte antigen
human leukocyte antigen
glucose metabolism
glycolysis
energy production
Casp-l
X
X
X
X
X
X
Casp-3
X
X
X
X
X
X
)
Known
substrate
74
Protein accession
number
ATP synthase, H+ transporting, mitochondrial Fl gi|13111901
complex, a subunit, isoform a precursor
carbonic anhydrase II
catalase
dihydrolipoamide dehydrogenase precursor
eosinophil preperoxidase
ferritin light subunit
Glutathione S-Transferase M2-3
glyceraldehyde-3-phosphate dehydrogenase
glyoxalase I
Horf6 A Chain B
lactoferrin precursor
leucine aminopeptidase
malate dehydrogenase (cytosolic)
malate dehydrogenase (mitochondial)
Myeloperoxidase
Myo-Inositol Monophosphatase Chain B
gi| 179780
gi| 179950
gi| 181575
gi|31183
gi|182516
gi|5822513
gi|31645
gi|5729842
gi|3318842
gi| 12083188
gi|4335941
gi|5174539
gi| 12804929
gi|494397
gi|2914661
)
Function Casp-1 Casp-3 Known
substrate
X
energy production
metabolism
metabolism
metabolism
metabolism
iron storage
protein folding
glycolysis
glucose metabolism
Peroxidase Enzyme
iron metabolism
metabolism
metabolism
metabolism
metabolism
metabolism
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
75
) )
Protein accession Function Casp-l Casp-3 Known
number substrate
ATP synthase, H+ transporting, mitochondrial FI gij13111901 X X
complex, Il subunit, isofonn a precursor energy production
carbonic anhydrase II gij179780 metabolism X
catalase gij179950 metabolism X
dihydrolipoamide dehydrogenase precursor gij181575 metabolism X
eosinophil preperoxidase gij31183 metabolism X
ferritin light subunit gij182516 iron storage X
Glutathione S-Transferase M2-3 gij5822513 protein folding X
glyceraldehyde-3-phosphate dehydrogenase gij31645 glycolysis X X
glyoxalase 1 gij5729842 glucose metabolism X
Horf6 A Chain B gij3318842 Peroxidase Enzyme X
lactoferrin precursor gij12083188 iron metabolism X
leucine aminopeptidase gij4335941 metabolism X
malate dehydrogenase (cytosolic) gij5174539 metabolism X
malate dehydrogenase (mi tochondial) gij12804929 metabolism X
Myeloperoxidase gij494397 metabolism X
Myo-Inositol Monophosphatase Chain B gij2914661 metabolism X
75
Protein
NADH cytochrome b5 reductase
pyruvate kinase
rho GDP dissociation inhibitor (GDI)
similar to catalase
transaldolase 1
Triosephosphate isomerase, glycolysis
accession number
gi|553254
gi|35505
gi|36038
gi|4557014
gi|5803187
gi| 136066
Tyrosine 3/tryptophan 5 -monooxygenase activation gi|80477445
protein, zeta
a-enolase gi(4503571
Channels, transporter and signaling
Annexin Hi
adenylate kinase 2 isoform b
ADP-ribosylation factor 4
Annexin A2
caspase-1
gi| 1421662
gi|7524346
gi|4502205
gi| 16306978
gi|2914146
Function
metabolism
glycolysis
signaling
metabolism
glucose metabolism
glycolysis
metabolism
Casp-1
X
X
X
X
X
glycolysis X
Casp-3 Known
substrate
X
X
X
membrane protein X
signaling X
signaling X
membrane protein X X
maturation of IL-ip and X X
apoptosis
76
")
Protein
NADH cytochrome b5 reductase
pyruvate kinase
rho GDP dissociation inhibitor (GD!)
similar to catalase
transaldolase 1
Triosephosphate isomerase , glycolysis
Tyrosine 3/tryptophan 5 -monooxygenase activation
protein, zeta
a-enolase
Channels, transporter and signaling
Annexin Iii
adenylate kinase 2 isoform b
ADP-ribosylation factor 4
AnnexinA2
caspase-l
accession
number
gil553254
gil35505
gil36038
gil4557014
gil5803187
gill36066
gil80477445
gil4503571
gil1421662
gil7524346
gil4502205
gil16306978
gil2914146
)
Function Casp-l Casp-3 Known
substrate
metabolism X
glycolysis X X
signaling X
metabolism X
glucose metabolism X
X glycolysis
metabolism X
glycolysis X
membrane protein X
signaling X
signaling X
membrane protein X X
maturation of IL-l ~ and X X
apoptosis
76
)
Protein accession
number
endoplasmic reticulum protein 29 isoform 1 gi|5803013
gi|56676393
precursor Rho GDP dissociation inhibitor p
globin p gi|26892090
GTP binding protein gi|4092054
guanine nucleotide-binding regulatory protein gi|183182
a-inhibitory subunit
migration-inducing gene 10 protein gi|41350401
p64 CLCP gi|895845
PA28 P gi|2136005
polypyrimidine tract-binding protein 1 a gi|4506243
PPPIA gi| 190281
proteasome a type, 8 isoform 1
protein PP4-X
gi|68303561
gi|189617
Rab5 gi|642532
)
Function
signaling
Casp-1
X
Casp-3 Known
substrate
signaling X
O2 transporter
signaling
signaling
signaling
chloride channel
proteasome activator
signaling
protein phosphatase
subunit, signaling
signaling
I a
Annexin A4, membrane
protein
ras-related small GTP
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
77
)
Protein
endoplasmic reticulum protein 29 isoform
precursor
Rho GDP dissociation inhibitor ~
globin ~
GTP binding protein
accession
number
gij5803013
gij56676393
gij26892090
gij4092054
guanine nuc1eotide-binding regulatory protein gij183182
a-inhibitory subunit
migration-inducing gene 10 protein
p64 CLCP
PA28 ~
polypyrimidine tract-binding protein 1 a
PPPIA
proteasome a type, 8 isoform 1
protein PP4-X
Rab5
gij413504û1
gij895845
gij2136005
gij4506243
gij190281
gij68303561
gij189617
gij642532
Function
signaling
signaling
O2 transporter
signaling
signaling
signaling
chloride channel
proteasome activator
signaling
protein phosphatase 1 a
subunit, signaling
signaling
Annexin A4, membrane
protein
ras-related small GTP
Casp-l
x
x
x
X
X
X
X
X
X
X
X
Casp-3
X
X
X
X
X
Known
substrate
X
')
77
')
Protein accession
number
Ran-specific GTPase-activating protein
serum albumin
SET translocation
gi|542991
gi|28592
gi|4506891
TRAP-1 gi| 1082886
Others
hypothetical protein LOC345651
PREDICTED: similar to POTE2A
unnamed protein product
gi|63055057
gi|51460457
gi|28590
)
Function
binding protein
signaling
steroid carrier
related to myeloid
leukemia
TNF receptor type 1
associated protein
X
X
X
Casp-1 Casp-3 Known
substrate
X
X
X
X
78
')
Protein
Ran-specific GTPase-activating protein
serum albumin
SET translocation
TRAP-l
Others
hypothetical protein LOC345651
PREDICTED: similar to POTE2A
unnamed protein product
accession
number
gil542991
gi128592
gij4506891
gil1082886
gil63055057
gil51460457
gil28590
Function Casp-l
binding protein
signaling X
steroid carrier
related to myeloid X
leukemia
TNF receptor type 1
associated protein
Casp-3
X
X
X
X
X
)
Known
substrate
78
Table 2 Cloning primers for PCR
cDNA and clone site
GAPDH (Xhol)
GAPDH (EcoRl)
a -enolase (Hindlll)
a -enolase (NOTI)
aldolase (Hindlll)
aldolase (NOTI)
a -enolase nested (Hindlll)
a -enolase nested (NOTI)
aldolase nested (Hindlll)
aldolase nested (NOTI)
TIM (HINDIII)
TIM (NOTI)
Pyruvate kinase (NOTI)
Pyruvate kinase (Xhol)
Primers
forward 5'-GCGGCTCGAGATGGGGAAGGTGAAGGTCGG-3*
reverse 5'-GCGGGAATCCTTACTCCTTGGAGGCCATGTGGG-3'
forward 5*-GCGGAAGCTTATGTCTATTCTCAAGATCCATGCC-3*
reverse 5'-GCGGGCGGCCGCTTACTTGGCCAAGGGG-3'
forward 5*-GGCCAAGCTTATGCCCTACCAATATCCAGC-3'
reverse 5'-GCGGGCGGCCGCTTAATAGGCGTGGTTAGAGACG-3'
forward 5'-GCGGAAGCTTCGGACAGTATCTGTGGGTACC-3'
reverse 5'-GCGGGCGGCCGCCGAGCTGCCTGAGCTGACACG-3'
forward 5'-GCGGAAGCTTGGGGTGCCTCAACCACACTCCG-3'
reverse S'-GCGGGCGGCCGCGCCCCGAGGAGGCGGCCTCC-S*
forward 5'-GGCGAAGCTTATGGCGCCCTCCAGGAAGTTCTTCG-3'
reverse 5'-GGCGGCGGCCGCTCATTGTTTGGCATTGATGATGTCC-3'
forward S'-GGCGGCGGCCGCATGTCGAAGCCCCATAGTGAAGCCGGG-S'
reverse 5*-GGCGCTCGAGTCACGGCACAGGAACAACACGCATGG-3'
") /
cDNA and clone site
GAPDH (Xhol)
GAPDH (EeoRl)
a ·enolase (HindlII)
a ·enolase (NO Tl)
aldolase (HindIlI)
aldolase (NO TI)
a ·enolase nested (HindlIl)
a ·enolase nested (NOTI)
aldolase nested (HindIlI)
aldolase nested (NOTI)
TIM (HINDlIl)
TlM(NOTI)
Pyruvate kinase (NOTI)
Pyruvate kinase (Xhol)
1 forward
1 reverse
1 forward
1 reverse
forward
1 reverse
1 forward
1 reverse
forward
1 reverse
forward
1 reverse
forward
1 reverse
Table 2 Cloning prim ers for PCR
Primers
5'·GCGGCTCGAGATGGGGAAGGTGAAGGTCGG·3'
5'·GCGGGAATCCTTACTCCTTGGAGGCCATGTGGG·3'
5'·GCGGAAGCTTATGTCTATTCTCAAGATCCATGCC·3'
5'·GCGGGCGGCCGCTTACTTGGCCAAGGGG·3'
5'·GGCCAAGCTTATGCCCTACCAATATCCAGC·3'
5'·GCGGGCGGCCGCTTAATAGGCGTGGTTAGAGACG-3'
5'·GCGGAAGCTTCGGACAGTATCTGTGGGTACC·3'
5'·GCGGGCGGCCGCCGAGCTGCCTGAGCTGACACG·3'
5'-GCGGAAGCTTGGGGTGCCTCAACCACACTCCG-3'
5 '·GCGGGCGGCCGCGCCCCGAGGAGGCGGCCTCC·3 ,
5 '·GGCGAAGCTTATGGCGCCCTCCAGGAAGTTCTTCG-3 ,
5'-GGCGGCGGCCGCTCATTGTTTGGCATTGATGATGTCC-3'
5'·GGCGGCGGCCGCATGTCGAAGCCCCATAGTGAAGCCGGG-3'
5'·GGCGCTCGAGTCACGGCACAGGAACAACACGCATGG-3'
)
79
Table 3 Primers for potential cleavage sites mutagenesis
cDNA and mutation site Primers
a -enolase
a -enolase
a -enolase
a -enolase
a -enolase
a -enolase
a -enolase
a -enolase
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
D136A
D136A
D238A
D238A
D266A
D266A
D286A
D286A
D144A
D144A
D166A
D166A
D189A
D189A
D198A
D198A
forward 5'-CCCCCTGTACCGCCACATCGCTGCCTTGGCTGGCAACTCTGAAGTCATCC-3'
reverse 5'-GGATGACTTCAGAGTTGCCAGCCAAGGCAGCGATGTGGCGGTACAGGGGG-3'
forward 5'-GGGAAAGCTGGCTACACTGCTAAGGTGGTCATCGG-3'
reverse 5'-CCGATGACCACCTTAGCAGTGTAGCCAGCTTTCCC3-3*
forward 5-'CTTCAAGTCTCCCGATGCCCCCAGCAGGTACATC-3'
reverse 5'-GATGTACCTGCTGGGGGCATCGGGAGACTTGAAG-3'
forward 5'-CAAGTCCTTCATCAAGGCCTACCCAGTGGTGTC-3'
reverse 5'-GACACCACTGGGTAGGCCTTGATGAAGGACTTG-3'
forward 5'-GGGTGTGAACCATGAGAAGTATGCCAACAGCCTCAAGATCATCAGC-3'
reverse 5'-GCTGATGATCTTGAGGCTGTTGGCATACTTCTCATGGTTCACACCC-3'
forward 5'-CCCTGGCCAAGGTC ATCC ATGCCAACTTTGGTATCGTGGAAGG-3'
reverse 5'-CCTTCCACGATACCAAAGTTGGCATGGATGACCTTGGCCAGGG-3'
forward 5'-CTGCCACCCAGAAGACTGTGGCTGGCCCCTCCGGGAAACTGTG-3'
reverse 5'-CACAGTTTCCCGGAGGGGCCAGCCACAGTCTTCTGGGTGGCAG-3'
forward 5'-CCCTCCGGGAAACTGTGGCGTGCTGGCCGCGGGGCTCTCCAGAAC-3'
reverse 5'-GTTCTGGAGAGCCCCGCGGCCAGCACGCCACAGTTTCCCGGAGGG-3'
.~ )
Table 3 Primers for potential cleavage sites mutagenesis
Primers cDNA and mutation site
a -enolase D136A forward 5'-CCCCCTGTACCGCCACATCGCTGCCTTGGCTGGCAACTCTGAAGTCATCC-3'
a -enolase D136A reverse 5'-GGATGACTTCAGAGTTGCCAGCCAAGGCAGCGATGTGGCGGTACAGGGGG-3'
a -enolase D238A forward 5'-GGGAAAGCTGGCTACACTGCTAAGGTGGTCATCGG-3'
a -enolase D238A reverse 5'-CCGATGACCACCTTAGCAGTGTAGCCAGCTTTCCC3-3'
a -enolase D266A forward 5-'CTTCAAGTCTCCCGATGCCCCCAGCAGGTACATC-3'
a -enolase D266A reverse 5'-GATGTACCTGCTGGGGGCATCGGGAGACTTGAAG-3'
a -enolase D286A forward 5'-CAAGTCCTTCATCAAGGCCTACCCAGTGGTGTC-3'
a -enolase D286A reverse 5'-GACACCACTGGGTAGGCCTTGATGAAGGACTTG-3'
GAPDH D144A 1 forward 5'-GGGTGTGAACCATGAGAAGTATGCCAACAGCCTCAAGATCATCAGC-3'
GAPDH D144A reverse 5'-GCTGATGATCTTGAGGCTGTTGGCATACTTCTCATGGTTCACACCC-3'
GAPDH D166A forward 5'-CCCTGGCCAAGGTCATCCATGCCAACTTTGGTATCGTGGAAGG-3 '
GAPDH D166A reverse 5'-CCTTCCACGATACCAAAGTTGGCATGGATGACCTTGGCCAGGG-3'
GAPDH D189A forward 5 '-CTGCCACCCAGAAGACTGTGGCTGGCCCCTCCGGGAAACTGTG-3 ,
GAPDH D189A reverse 5'-CACAGTTTCCCGGAGGGGCCAGCCACAGTCTTCTGGGTGGCAG-3'
GAPDH D198A forward 5'-CCCTCCGGGAAACTGTGGCGTGCTGGCCGCGGGGCTCTCCAGAAC-3 '
GAPDH D198A reverse 5'-GTTCTGGAGAGCCCCGCGGCCAGCACGCCACAGTTTCCCGGAGGG-3 '
80
REFERENCE
1. Alam, A., Cohen, L.Y., Aouad, S., and Sekaly, R.P. (1999). Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells. The Journal of experimental medicine 190,1879-1890.
2. Andrei, C , Margiocco, P., Poggi, A., Lotti, L.V., Torrisi, M.R., and Rubartelli, A. (2004). Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proceedings of the National Academy of Sciences of the United States of America 101,9745-9750.
3. Arai, J., Katai, N., Kuida, K., Kikuchi, T., and Yoshimura, N. (2006). Decreased retinal neuronal cell death in caspase-1 knockout mice. Japanese journal of ophthalmology 50, 417-425.
4. Aries, A., Paradis, P., Lefebvre, C , Schwartz, R.J., and Nemer, M. (2004). Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proceedings of the National Academy of Sciences of the United States of America 101,6975-6980.
5. Atkinson, E.A., Barry, M., Darmon, A.J., Shostak, I., Turner, P.C., Moyer, R.W., and Bleackley, R.C. (1998). Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. The Journal of biological chemistry 273,21261-21266.
6. Barrei ro, E., Gea, J., Di Falco, M., Kriazhev, L., James, S., and Hussain, S.N. (2005). Protein carbonyl formation in the diaphragm. American journal of respiratory cell and molecular biology 32,9-17.
7. Beyaert, R., Kidd, V.J., Cornells, S., Van de Craen, M., Denecker, G., Lahti, J.M., Gururajan, R., Vandenabeele, P., and Fiers, W. (1997). Cleavage of PITSLRE kinases by ICE/CASP-1 and CPP32/CASP-3 during apoptosis induced by tumor necrosis factor. The Journal of biological chemistry 272,11694-11697.
8. Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci, J.E., Edris, W.A., Sutherlin, D.P., Green, D.R., et al (2003). A unified model for apical caspase activation. Molecular cell 11,529-541.
9. Boudreau, N., Sympson, C.J., Werb, Z., and Bissell, M.J. (1995). Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267, 891-893.
10. Cavaillon, J.M., Adib-Conquy, M., Fitting, C , Adrie, C , and Payen, D. (2003). Cytokine cascade in sepsis. Scandinavian journal of infectious diseases 35,535-544.
11. Chen, Z., Naito, M., Mashima, T., and Tsuruo, T. (1996). Activation of actin-cleavable interleukin 1 beta-converting enzyme (ICE) family protease CPP-32 during chemotherapeutic agent-induced apoptosis in ovarian carcinoma cells. Cancer research 55,5224-5229.
12. Chi, M.M., Pingsterhaus, J., Carayannopoulos, M., and Moley, K.H. (2000). Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine
82
REFERENCE
1. Alam, A., Cohen, L. Y., Aouad, S., and Sekaly, RP. (1999). Early activation of caspases
during T lymphocyte stimulation results in selective substrate c1eavage in nonapoptotic cells. The Journal of experimental medicine 190,1879-1890.
2. Andrei, C, Margiocco, P., Poggi, A., Lotti, L. V., Torrisi, M.R, and Rubartelli, A. (2004). Phospholipases C and A2 control Iysosome-mediated IL-l beta secretion: Implications for inflammatory processes. Proceedings of the National Academy of Sciences ofthe United States of America 101, 9745-9750.
3. Arai, J., Katai, N., Kuida, K., Kikuchi, T., and Yoshimura, N. (2006). Decreased retinal
neuronal cell death in caspase-l knockout mice. Japanese journal of ophthalmology 50,
417-425. 4. Aries, A., Paradis, P., Lefebvre, C, Schwartz, RJ., and Nemer, M. (2004). Essential
role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proceedings of the National Academy of Sciences of the United States of America 101, 6975-6980.
5. Atkinson, E.A., Barry, M., Darmon, A.J., Shostak, 1., Turner, P.C, Moyer, R. W., and Bleackley, RC (1998). Cytotoxic T Iymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. The Journal of biological
chemistry 273, 21261-21266.
6. Barreiro, E., Gea, J., Di Falco, M., Kriazhev, L., James, S., and Hussain, S.N. (2005). Protein carbonyl formation in the diaphragm. American journal of respiratory cell and
molecular biology 32, 9-17.
7. Beyaert, R., Kidd, V.J., Cornelis, S., Van de Craen, M., Denecker, G., Lahti, J.M., Gururajan, R, Vandenabeele, P., and Fiers, W. (1997). Oeavage of PITSLRE kinases by ICE/CASP-l and CPP32/CASP-3 during apoptosis induced bytumor necrosis faètor. The Journal of biological chemistry 272, 11694-11697.
8. Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci,
J.E., Edris, W.A., Sutherlin, D.P., Green, D.R, et al (2003). A unified model for apical caspase activation. Molecular cellll, 529-541.
9. Boudreau, N., Sympson, CJ., Werb, Z., and Bissell, M.J. (1995). Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267, 891-893.
10. Cavaillon, J.M., Adib-Conquy, M., Fitting, C, Adrie, C, and Payen, D. (2003). Cytokine cascade in sepsis. Scandinavian journal of infectious diseases 35, 535-544.
11. Chen, Z., Naito, M., Mashima, T., and Tsuruo, T. (1996). Activation of actin-c1eavable interleukin Ibeta-converting enzyme (ICE) family protease CPP-32 during
chemotherapeutic agent-induced apoptosis in ovarian carcinoma cells. Cancer research 56,5224-5229.
12. Chi, M.M., Pingsterhaus, J., Carayannopoulos, M., and Moley, K.H. (2000). Decreased
glucose transporter expression triggers BAX-dependent apoptosis in the murine
82
blastocyst. The Journal of biological chemistry 275,40252-40257.
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M.E., Offringa, R., and Krammer, P.H. (1997). Oeavage of FLICE (caspase-8) by
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74. Monack, D.M., Hersh, D., Ghori, N., Bouley, D., Zychlinsky, A., and Falkow, S. (2000).
Salmonella exploits caspase-l to colonize Peyer's patches in a murine typhoid model.
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90
APPENDIX I Caspase-1 substrate: GATA-4
GATA-4 belongs to the GATA family of zinc finger transcription factors. It is expressed
in developing cardiac cells and responsible for cardiac differentiation during early
embryonic development (Heikinheimo et al., 1994). In adults, GATA-4 continues to be
expressed in cardiac myocytes (Pikkarainen et al., 2004). It has been proposed that
GATA-4 is required for the adaptive response of cardiomyocytes (Suzuki et al., 2004).
GATA-4 was implicated in the protection of cardiac myocytes from apoptosis induced by
antitumor treatment (Aries et al., 2004; Kim et al., 2003). The function of GATA-4 in cell
survival was also supported by the fact that over expression of GATA-4 increased
differentiation of cardiomyocytes, while inhibition of GATA-4 by an antisense strategy
prevented cardiomyocyte differentiation and triggered extensive apoptosis (Grepin et al.,
1997; Grepin et al., 1995). The action of GATA-4 in cell survival was suggested to occur
through the the upregulation of Bcl-XL transcription by GATA-4 (Aries et al., 2004).
It has been shown that doxorubicin treatment induces apoptosis in cultured
cardiomyocytes and GATA-4 is rapidly depleted during this process (Aries et al., 2004;
Kim et al., 2003). To identify the factors that are responsible for the GATA-4 depletion
during apoptosis, Dr. Mona Nemer' lab collaborated with our lab. Since there exist
potential caspase-1 and -3 cleavage sites in the GATA-4 protein, we first tested the
cleavage of GATA-4 by casapses-1, and - 3 . We in vitro transcribed and translated 35S
labeled rat GATA-4 and digested with recombinant caspase-1 and - 3 . Our results show
that GATA-4 is cleaved by caspase-1 in vitro, but not by caspase-3 (Appendix I figure 1
A). Site-directed mutagenesis of potential cleavage sites of GATA-4 revealed that a
combination of two point mutations D168A and D230A blocked caspase-1 cleavage
(Appendix I figure 1 B, C). The cleavage sequences are YMAD168iV and WRRD230|G,
91
APPENDIX 1 Caspase-l substrate: GA TA-4
GATA-4 belongs to the GATA family of zinc finger transcription factors. It is expressed
in developing cardiac cells and responsible for cardiac differentiation during early
embryonic development (Heikinheimo et al., 1994). In adults, GATA-4 continues to be
expressed in cardiac myocytes (pikkarainen et al., 2004). It has been proposed that
GATA-4 is required for the adaptive response of cardiomyocytes (Suzuki et al., 2004).
GATA-4 was implicated in the protection of cardiac myocytes from apoptosis induced by
antitumor treatment (Aries et al., 2004; Kim et al., 2003). The function of GATA-4 in cell
survival was also supported by the fact that over expression of GATA-4 increased
differentiation of cardiomyocytes, while inhibition of GATA-4 by an antisense strategy
prevented cardiomyocyte differentiation and triggered extensive apoptosis (Grepin et al.,
1997; Grepin et al., 1995). The action of GATA-4 in cell survival was suggested to occur
through the the \lpregulation of Bc1-XL transcription by GATA-4 (Aries et al., 2004).
It has been shown that doxorubicin treatment induces apoptosis in cultured
cardiomyocytes and GATA-4 is rapidly depleted during this process (Aries et al., 2004;
Kim et al., 2003). To identify the factors that are responsible for the GATA-4 depletion
during apoptosis, Dr. Mona Nemer' lab collaborated with our labo Since there exist
potential caspase-l and -3 cleavage sites in the GATA-4 protein, we first tested the
c1eavage of GATA-4 by casapses-l, and -3. We in vitro transcribed and translated 35S
labeled rat GATA-4 and digested with recombinant caspase-l and -3. Our results show
that GATA-4 is c1eaved by caspase-l in vitro, but not by caspase-3 (Appendix l figure 1
A). Site-directed mutagenesis of potential c1eavage sites of GATA-4 revealed that a
combination of two point mutations D168A and D230A blocked caspase-l c1eavage
(Appendix 1 figure 1 B, C). The cleavage sequences are YMAD168JV and WRRD230JG,
91
which fit the criteria of a bulky or hydrophobic amino acid in the P4 position of a
caspase-1 substrate, and a small non-charged amino acid in the PI ' position. Alignment
of the GATA-4 sequences from different species showed that the cleavage site
WRRD230|G is conserved in all the species examined, while the cleavage site
YMAD168,|,V is conserved only in mammals (Appendix I figure 1 D). Structurally,
GATA-4 contains an N-terminal and a possible C-terminal transcriptional activation
domains, as well as two adjacent zinc fingers in the middle (Appendix I figure 2).
Caspase-1 cleaves GATA-4 at YMAD168jV removing its N-terminal transcriptional
activation domain and at WRRD230|G breaking one of its two zinc finger domains.
Therefore, we hypothesize that the cleavage of GATA-4 by caspase-1 causes its
inactivation.
Dr. Nemer and her colleagues have observed that GATA-4 was rapidly degraded 3 hours
after doxorubicin treatment of cultured cardiomyocytes (Aries et al., 2004). Cleavage
products of GATA-4 were not detected by western blot. So, for future work, we need to
confirm the in vivo cleavage of GATA-4 by caspase-1. Since it has been reported that
some caspase substrates are degraded by the Ubiquitin/proteosome pathway (Demontis et
al., 2006; Ditzel et al., 2003; Du et al., 2005), in a next step, we can test the possibility of
GATA-4 degradation by this system. To do this, we would first inhibit the proteosome
using the proteosome inhibitor LLnL, we would then expect the visualization of GATA-4
cleavage after Doxorubicin treatment. After the in vivo cleavage of GATA-4 by caspase-1
is confirmed, we will investigate the relevance of the GATA-4 cleavage on cell survival.
92
wbieh fit the criteria of a bulky or hydrophobie amino aeid in the P4 position of a
caspase-l substrate, and a small non-charged ami no acid in the Pl' position. Alignment
of the GATA-4 sequences from different species showed that the c1eavage site
WRRD230 L G is conserved in aIl the species exarnined, while the c1eavage site
YMADl68 L V is conserved only in mammals (Appendix 1 figure 1 D). Structurally,
GATA-4 contains an N-terminal and a possible C-terminal transcriptional activation
dornains, as weIl as two adjacent zinc fingers in the rniddle (Appendix 1 figure 2).
Caspase-l c1eaves GATA-4 at YMADl68 LV rernoving its N-terminal transcriptional
activation domain and at WRRD230 LG breaking one of its two zinc finger dornains.
Therefore, we hypothesize that the c1eavage of GATA-4 by caspase-l causes its
inactivation.
Dr. Nemer and her coUeagues have observed that GATA-4 was rapidly degraded 3 hours
after doxorubicin treatment of cultured cardiomyocytes (Aries et al., 2004). Cleavage
products of GATA-4 were not detected by western blot. So, for future work, we need to
confirm the in vivo c1eavage of GATA-4 by caspase-l. Since it has been reported that
sorne caspase substrates are degraded by the Ubiquitin/proteosorne pathway (Demontis et
al., 2006; Ditzel et al., 2003; Du et al., 2005), in a next step, we can test the possibility of
GATA-4 degradation by tbis system. To do this, we would first inhibit the proteosome
using the proteosorne inhibitor LLnL, we would then expect the visualization of GATA-4
c1eavage after Doxorubicin treatment. Mter the in vivo c1eavage of GATA-4 by caspase-l
is confirmed, we will investigate the relevance of the GATA-4 c1eavage on ceU survival.
92
Appendix figure 1
A
I
100 75
50
37
25
20
15
1 j? > «? £ £ &
-™*
ai^Hk -n̂ Mtei ^^^H^
~ '̂BP
-
GATA-4 ITT
B
Rat GATA-4 YMAD»«8V WRRD230G
10-
c
"asp-1 7 5 -
5 0 - ^
3 7 -
2 5 -
2 0 -
{+ 18.5 K D a ^
i 25.3 KDa ^ r<i • ** 48.4KDa
wt D168A
+ - +
• !
D168A D230A D230A
+ - +
• j# i fc
GATA-4 ITT
93
~
'b ~ 'i" " Appendix figure 1 8
.5 l- i-:§ ü çJ
A 100 -
75
50 -. ,.. ... 37
25
20
15
B
RatGATA-4
c wt
Casp-l - + 75-
50-~
37-
25-20-
10-
• ..... GATA-4ITT
YMAD168V WRRD230G
48AKDa
D168A
+
GATA-4ITT
D230A
+
Dl68A D230A
+
93
Appendix figure 1 D
i human mouse rat chick
SPYPA SPYPA SPYPA SPYPA
VGASWAAAAAASAGPFDSPVLHS VGASWAAAAAASAGPFDSPVLHS VGASWAAAAAASAGPFDSPVLHS
EMATTWTSSPFDSPMLHN
i human mouse rat chick
RHPNLDMFDDFSEGRECVNCGAMSTPL RHPNLDMFDDFSEGRECVNCGAMSTPL RHPNLDMFDDFSEGRECVNCGAMSTPL RHANIEFFDDYSEGRECVNCGAMSTPL
GT GT GT GT
Appendix figure 1
D
human mouse rat chick
human mouse rat chick
SPYPA SPYPA SPYPA SPYPA
VGASWAAAAAASAGPFDSPVLHS VGASWAAAAAASAGPFDSPVLHS VGASWAAAAAASAGPFDSPVLHS -----EMATTWTSSPFDSPMLHN
RHPNLDMFDDFSEGRECVNCGAMSTPL RHPNLDMFDDFSEGRECVNCGAMSTPL RHPNLDMFDDFSEGRECVNCGAMSTPL RHANIEFFDDYSEGRECVNCGAMSTPL
GT GT GT GT
94
Appendix figure 1 Caspase-1 substrate GATA-4
(A) In vitro cleavage of ITT GATA-4 by caspase-1 and -3
(B) Analysis of GATA-4 cleavage sites
(C) Blockage of caspase-1 cleaving GATA-4 mutant (D168A D230A)
95
Appendix figure 1 Caspase-l substrate GATA-4
(A) ln vitro cleavage oflTT GATA-4 by caspase-l and-3
(B) Analysis of GATA-4 cleavage sites
(C) Blockage ofcaspase-l cleaving GATA-4 mutant (D168A D230A)
95
Appendix Figure 2
168 230
Transcriptional activation
N-tenn Zn C-torm Zn
Cofactor DNA& binding Cofactor
binding
Transcriptional activation (?)
Muelaar localization
Appendix Figure 2 Functional Structure of GATA-4 (Adapted from Pikkarainen el., 2004)
GATA-4 contains an N-terminal and a possible C-terminal transcriptional activation
domains, as well as two adjacent zinc fingers in the middle. Caspase-1 cleaves GATA-4
at YMAD168 I V removing its N-terminal transcriptional activation domain and at
WRRD230 \ G breaking one of its two zinc finger domains.
96
Appendix Figure 2
168 230
N.-nn Zn C .. rm Zn --
Nue/., locIIttz.tlon
Appendix Figure 2 Functional Structure ofGATA-4 (Adapted from Pikkarainen el., 2004)
GATA-4 contains an N-terminal and a possible C-terminal transcriptional activation
domains, as weIl as two adjacent zinc fingers in the middle. Caspase-l c1eaves GATA-4
at YMAD168 ~ V removing its N-terminal transcriptional activation domain and at
WRRD230 ~ G breaking one of its two zinc finger domains.
96
APPENDIX II
97
APPENDIXII
97
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