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- 2005: ANR «Maladies Rares» JP di Rago (yeast mitochondria)/M Blondel (yeast chemobiology)
- 2008: LM Steinmetz (yeast chemogenomics)
- 2011: FRM «Régulations Métaboliques»
A Roetig/JP di Rago/M Blondel/A Delahodde/G Dujardin
- 2014: AFM «Projet Stratégique»
A Roetig/JP di Rago/M Blondel/A Delahodde/G Dujardin/V Procaccio
LM Steinmetz/ML Jung
Yeast-based chemobiological approaches of
mitochondrial diseases
Marc Blondel ([email protected])
Inserm UMR1078, Brest, France
Chemobiology at happy hour: yeast models for
human diseases
Yeast as a model for human diseases
Marc Blondel ([email protected])
Inserm UMR1078, Brest, France
Marc Blondel,
Cécile Voisset, CR1, Inserm
Gaëlle Friocourt, CR1, Inserm
Déborah Tribouillard-Tanvier, CR2, Inserm
Olivier Billant, PhD, MRES
Maria-José Lista, PhD, La Ligue/Région
Marie-Astrid Contesse, IE, CDD UBO
Justine Evrard, IE, CDD Inserm
Nadège Loaëc, IE, CDD Inserm
Hélène Simon, AJT, UBO
Alice Léon, stage M2, UBO
yeast is an eukaryote as easy to handle as a prokaryote
cellular mechanisms & key players involved in most human diseases are conserved from yeast to humans therefore it is possible to create yeast models for human diseases… STARTING POINT: creating yeast models exhibiting a phenotype relevant for the pathology of interest
searching for modifiers + or - by screening:
• small MW compounds (identification of drug candidates) • genes/cDNA (identification of mechanisms)
validation of these modifiers (drugs, genes…): human cells, animal models… drugs are used for reverse screening: search for intracellular targets of drugs (identification of mechanisms)
drugs can be used as tools for basic research (chemical genetics)
yeast: a robust and versatile system to model human diseases
proofs of principle…
Yeast models for prion-based diseases (Bach et al. Nature Biotechnology 2003)
ex. of yeast-based drug screening
Yeast models for Huntington disease (Giorgini et al. Nature Genetics 2005)
ex. of yeast-based genetic screening
an example of a drug screening based on a yeast model for prion diseases:
active in vivo against
yeast prions
active ex vivo against
mammalian prion PrPSc
6-aminophenanthridine (6AP) & guanabenz (GA) are active against yeast &
mammalian prions both ex vivo & in vivo
active in vivo in a murine model for
prion-based diseases (collaboration
V. Beringue & H. Laude, INRA Jouy)
prion-controlling mechanisms are conserved from yeast to mammals
we identified the conserved target of 6AP & GA:
the protein folding activity of the ribosome (PFAR)
Brancusi
modelling human mitochondrial diseases in budding yeast:
from deciphering of physiopathological mechanisms
to isolation of candidate drugs:
a yeast-based model for the NARP syndrome
Marc Blondel
Inserm UMR1078
Faculté de Médecine et
des Sciences de la Santé
BREST
Jean-Paul di Rago
Institut de Biochimie et
Génétique Cellulaires
CNRS/Université Bordeaux2
UMR5095
BORDEAUX
a partnership between:
funded by ANR «Maladies Rares» 2006-8, AFM 2008-13 & FRM 2011-12
& in collab. with Lars Steinmetz,
EMBL Heidelberg, Germany
& Stanford University, USA
- Kucharczyk et al, 2009
- Kucharczyk et al, 2010
- Couplan et al, 2011
- Aiyar et al, 2014
NARP syndrome: a devastating mitochondrial disease
Neuropathy Ataxia Retinitis Pigmentosum
a maternally inherited mitochondrial disease (mutation within the mt DNA)
due to mutations in the mitochondrial ATP6 gene encoding a subunit of ATP
synthase, which is essential for the production of ATP by oxydative phosphorylation
(OXPHOS)
ADP ATP
F0
F1
H+ H+
O2
ATP synthase
respiratory
chain
Sous-unité
c
H+
H+
subunit a
(stator)
subunit c
(rotor) H2O
mitochondria =
cell powerhouse
(prod. ATP by OXPHOS)
reduction of O2 in H2O:
creation of a H+ gradient
used by ATP synthase
to produce ATP from ADP
ATP synthase =
nanomolecular engine,
fuel = H+ gradient
ATP synthase: a nano-molecular engine
NARP mutations:
lie in the stator, at the
interface with the rotor
Neuropathy Ataxia Retinitis Pigmentosum
a genetic disease: maternally inherited mitochondrial disease
due to mutations in the mitochondrial ATP6 gene encoding a subunit of the
stator of ATP synthase, these mutations block the rotor rotation
( like « a fly in the ointment »)
ADP ATP
F0
F1
H+ H+
O2
ATP synthase
respiratory
chain
Sous-unité
c
H+
H+
subunit a
(stator)
subunit c
(rotor)
deficiency in ATP production NARP syndrome
NARP syndrome: a devastating mitochondrial disease
like all eukaryotic cells, yeast cells do contain mitochondria (that have their own
mitochondrial genome) which are very similar to their human counterpart
why yeast
for modelling human mitochondrial diseases (myopathies, NARP, etc.)?
Cytosol
OM
IMS
IM
Matrix
14 subunits
Atp6p, Atp8p
ASSEMBLY
Nucleus
14 subunits
TOM
TIM
4
6 8
h
a
g
d e
d
9 9 9 9 9
oscp
i,f
b b
a
Human
mtDNA
ATP6 ATP8
Cytosol
OM
IMS
IM
Matrix
18 subunits
Atp6p, Atp8p, Atp9p
Nucleus
18 subunits
TOM
TIM
F1 : Atp11p,
Atp12p, Fmc1p
Fo : Atp23p,
Atp10p,Atp25
Expression of ATP6,
ATP8, ATP9
Nca1, Nca2, Nca3, Aep1,
Aep2, Aep3, Atp22, Atp25
4
6 8
h
a
g
d e
d
9 9 9 9 9
oscp
i,f
b b
a
ATP6
ATP8
ATP9
S.cerevisiae
mtDNA
ASSEMBLY
mitochondrial functions are well conserved
from yeast to human, in particular ATP synthase
like all eukaryotic cells, yeast cells do contain mitochondria (that have their own
mitochondrial genome) which are very similar to their human counterpart
budding yeast can survive w/o respiration (using fermentation to produce ATP)
why yeast
for modelling human mitochondrial diseases (myopathies, NARP etc.)?
GLUCOSE CO2 + H20
ADP+Pi ATP
Oxidative
Phosphorylation
Fermentation
ADP+Pi ATP GLUCOSE CO2 + EtOH
WT
yeast
respiratory
mutant
fermentable medium
(glucose)
respiratory medium
(glycerol)
budding yeast can survive w/o respiration (using fermentation to produce ATP): fermentation > very rapid but low yield / respiration > very slow but high yield
hence budding yeast can be used to perform pharmacological screening on NARP
yeast is one of the very few eucaryotes for which site-directed mutagenesis of the mitoch.
genome is possible (using a biolistic technique): introd. mut. synonymous to NARP mut.
like all eukaryotic cells, yeast cells do contain mitochondria (that have their own
mitochondrial genome) which are very similar to their human counterpart
budding yeast can survive w/o respiration (using fermentation to produce ATP)
why yeast
for modelling human mitochondrial diseases (myopathies, NARP etc.)?
ATP6
ATP8
ATP9
biolistic
site-directed
mutagenesis
JP di Rago
T8993G
T8993C
T9176G
T9176C
T8551C
NARP/MILS
S.cerevisiae
mtDNA
respiratory growth defect of yeast NARP mutants at 37°C
normal growth on glucose
ATP ↔ Fermentation
glucose
drops of serial dilutions of yeast cell liquid cultures
mutat. within
the yeast ATP6
gene
synonymous
to mutations
found in
NARP patients
T8993G
T8993C
T9176G
T9176C
T8851C
Wild type
fmc1
glycerol
growth defect on glycerol
ATP ↔ Respiration
ATP synthesis
% of WT
100
74
<5
6
8
7
50
perfect correlation with
severity of the mutations
in NARP patients!
perfect correlation > 1st validation of the yeast model for NARP…
genetic screening (JP di Rago & M Rigoulet)
ODC1 was isolated as a multicopy suppressor of NARP mutations in yeast (JP di Rago & M Rigoulet)
Odc1p
Porin
western-blot Glucose Glycerol
atp synthase
mutant
atp synthase
mutant + oe ODC1
ODC1 encodes oxodicarboxylate carrier
NARP/fmc1Δ
strain
in glucose
filters one drug
per filter
incubation
5-7 days at 35°C
active compound
negative control (DMSO)
NARP/fmc1Δ
strain spread
on respiratory
solid medium
(glycerol)
drug screening (E Couplan, JP di Rago & M Blondel)
compounds from chemical libraries are tested for their ability
to suppress the growth defect of yeast NARP mutants
pos. control = Di-Hydro Lipoïc Acid (DHLA) currently in clinic to treat
mitochondrial encephalopathies: 2nd validation of the yeast assay!
>12,000 molecules screened (including the Prestwick C.L.): ~20 active compounds
positive
control (DHLA)
HS
SH
OH
O
currently tested
in clinic to treat
mitochondrial
encephalopathies
example of an active compound: chlorhexidine (CH),
a compound able to suppress the respiratory phenotype
of NARP and ATP synthase mutants in yeast
T89
93
G
T91
76
G
Wil
d t
yp
e
T88
51
C
fmc1
NARP mut.
DMSO
CH 2 µM
…CH suppresses respiratory growth
defect when added directly in the medium…
& its effect is allele specific
CH suppresses respiratory
growth defect on solid medium…
3rd validation of the yeast assay: activity of DHLA & CH on NARP cybrids
NARPJCP239 cybrids mtDNA T8993G
cybrids = fusion between:
- rho0 osteosarcome (thus a cell line w/o mtDNA)
- platelets from NARP patients
(w/o nucleus & containing mtDNA T8993G )
hence resulting cybrids are cell lines containing NARP mitochondria
growth of NARPJCP239 cybrids in glucose similar to WT cybrids
because it relies on glycolysis (like most of tumoral cell lines)
w/o glucose: cybrids are forced to rely on oxidative phosphorylation
for their growth: pronounced growth defect of NARPJCP239 cybrids
compared to WT control cybrids (similar to the yeast situation)
drugs can be tested on primary cultures of fibroblasts from patients:
difficult to handle & no clear phenotype… thus we used another system:
effect of CH on the growth of NARP cybrids in a medium deprived of glucose
CH DHLA 200 µM
CH & DHLA also active on NARP cybrids: validation of the yeast assay & of CH
[CH] nM
Perc
enta
ge o
f gr
ow
th
0
100
200
DHLA 0 12.5 25 50 80
200 µM
CH acts on mitochondria in yeast V
ox (
nA
tO2.m
in-1
.mg
-1)
0
100
200
300
400
500
600
EtOH TET CCCP
fmc1 + DMSO fmc1 + CH
FMC1+
CH increases respiration
rate of mutant cells…
fmc1
+
DM
SO
0.5 µm
V
N
0.2 µm0.2 µm
0.2 µm0.2 µm
m
m m
m
ATP synthase mutants:
accumulation of inclusion bodies
within mitochondria
& disapearance of cristae
fmc1
+
CH
0.5 µm
V
N
0.2 µm0.2 µm
0.2 µm0.2 µm
m
ATP synthase mutants + CH:
disapearance of inclusion bodies
within mitochondria
& reappearance of cristae
Cyt b
Atp9
Atp1
Cox2
Porin
Actin F
MC
1+
-
fmc1
+ - CH
ATP Synthase
subunits
(Complexe V)
Complex IV
subunit
Complex III
subunit
outer membr.
protein
CH restores levels
of several proteins of the
mitochondrial OXPHOS
pathway
determination of all genes whose expression is significantly affected
in ATP synthase mutant / WT strains & of CH effect on these genes…
global transcriptomic analysis by tilling arrays (R. Aiyar & L. Steinmetz, EMBL,
Heidelberg, Germany & Stanford University, USA)
most of the nuclear genes affected (down or upregulated)
in ATP synthase mutant / WT
are nuclear genes encoding mitochondrial proteins (~50)…
global transcriptomic analysis by tilling arrays (R. Aiyar & L. Steinmetz, EMBL,
Heidelberg, Germany & Stanford University, USA)
CH partially or totally restores expression of most of
these genes
QCR9 gene is the only one to be down-regulated in
ATP synthase mutants & very rapidly (after a few min.)
overexpressed when CH is added… thus QCR9 could
be a critical target for CH activity…
& indeed QCR9 overexpression partially suppresses the
respiratory growth defect of ATP synthase mutants…
& QCR9
conserved in
humans…
Sodium pyrithione (NaPT): another active compound
DMSO
CH
NaPT
DHLA
fmc1Δ @ 36°C
NaPT suppresses the respiratory growth defect of
an ATPsynthase mutant at 36°C
in a dose-dependent manner
quantity
0
20
40
60
80
100
120
140
160
180
200
DMSO 100 µM DHLA
20 nM C7
40 nM C7
60 nM C7
80 nM C7
100 nM C7
25 nM C1
Cell viability after a 6 days treatmentJC239 (NARP)
JC213 (WT)
NaPT is active on NARP cybrids
& has no effect on WT cybrids
CH
sodium pyrithione (NaPT)
NaPT mode of action? determination of its cellular target(s)
using chemogenomics (collab. L. Steinmetz, JP di Rago & M. van der Laan)
chemical genomics in yeast: finding drug targets via gene dosage G
row
th
Normal
Haploinsufficiency
Deletion
sensitivity
Inhibited
by drug
drug
method: take drug of interest at a concentration that partially inhibits cell growth
* when one copy of its target is deleted (haploinsufficiency), growth should be hypersensitive to the drug = deletion
sensitivity
* when its target is overexpressed from a plasmid, growth defect should be suppressed = multicopy suppression
Multicopy suppression
Overexpression
B. StOnge, Stanford
Haploinsufficiency (X axis) for every single yeast gene is
plotted as a function of Multicopy suppression (Y axis):
the most interesting genes are in the top right
Tim17, a component of the mitochondrial TIM
complex was identified as a putative target of NaPT
chemical genomics in yeast : identification of Tim17 as a target of NaPT collab. Bob StOnge (Stanford) & Lars Steinmetz (Heidelberg)
B. StOnge, Stanford
haploinsufficiency
overexpression
TIM
17 D
ele
tion
Se
nsitiv
ity
1000+ compounds (by date screened)
NaPT
this level of Tim17 sensitivity is by far the highest
observed among >1,000 compounds screened at Stanford.
TOM and TIM allow import within the mitochondria of
mitochondrial proteins synthesized in the cytoplasm
Tim17p is part of TIM23 complex, a mitochondrial
presequence translocase
NaPT affects import
through this TIM23
supercomplex
by favouring the
lateral sorting
what does Tim17 do?
- Tim17 is an essential component of the TIM23 supercomplex, which mediates mitochondrial import of all
presequence-carrying proteins, targeting them either to the matrix or inner membrane (lateral sorting) depending
on the substrate’s destination.
- Therefore the TIM23 complex exists in two forms: one that allows matrix import and the other (which contains
Tim21) responsible for the lateral sorting (that allows insertion in the inner membrane of proteins of the various
complexes of the respiratory chain).
TIML23 complex =
Tim23, Tim17,
Tim50 and Tim21
overexpression of
Tim21p also favours
the lateral sorting
overexpression of TIM21 which favours lateral sorting mimics the effect of C7
in yeast…
WT fmc1Δ R. Aiyar, Heidelberg
Re
sp
ira
tory
overexpression of TIM21 which favours lateral sorting mimics the effect of C7
in yeast… and also in NARP cybrids…
ODC1 was isolated as a multicopy suppressor of NARP mutations in yeast (JP di Rago & M Rigoulet)
Odc1p
Porin
western-blot Glucose Glycerol
atp synthase
mutant
atp synthase
mutant + oe ODC1
ODC1 gene and oleate (OA) effect on yeast and NARP cybrids
ODC1 gene and oleate (OA) effects on yeast and NARP cybrids
ODC1 was isolated as a multicopy suppressor gene of NARP mutations in yeast,
oleate (OA) is known to lead to overexpression of ODC1 gene in yeast,
therefore we tested the activity of OA in the yeast model for NARP: OA is active,
ODC1 gene is conserved in humans, thus we tested OA in NARP cybrids:
oleate (OA) is active in cybrids
Oleate
OH
O
conclusions:
- 4 drugs (CH, DHLA, NaPT & OA) active in both yeast & human models for NARP
(& DHLA tested in clinic on patients):
validation of the yeast model for NARP & of CH, NaPT & OA
- CH mode of action: increases respiration an restores mitochondrial morphology,
its effect may be mediated by QCR9 gene (for which CH induces an overexpres.)
- this yeast-based model for NARP = proof of principle
this type of method could be used for many other mitochondrial diseases
(myopathies, etc): within the Theramito consortium we are currently developing
yeast-based models for five other human mitochondrial diseases… both in
S. cerevisiae and in S. pombe…
… and many others can be envisioned: creation of a « target library »
(>100 ≠ mitoch. disorders, individually rare but altogether not neglectable)
- NaPT mode of action: potentially by favouring lateral sorting at TIM level,
highlighting the mitochondrial import as a potential relevant therapeutic target
the Theramito consortium: 8 teams developing an integrated (yeasts, nematode, cells from
patients, cybrids) approach for mitochondrial diseases (6 dif. diseases)
- 3 yeast labs:
- JP di Rago (IBGC, Bordeaux)
- G. Dujardin (CGM, Gif/Yvette)
- M. Blondel (Inserm UMR1078, Brest)
- 1 nematode lab:
- A. Delahodde (IGM, Orsay)
- 2 labs having access to patients and patients’ cells:
- A. Roetig (Hôp. Necker, Paris)
- V. Procaccio (CHU, Angers)
- 1 SME specialized in medicinal chemistry:
- Prestwick Chemical (Ilkirch)
- 1 lab of chemogenomics:
- L. M. Steinmetz (EMBL, Heidelberg)
dir.: Agnès Roetig