lys

Two strategies for protein degradationTwo strategies for protein degradation

1) Send the protein to a degradative compartment

2) selective degradation of individual molecules

Cellular protein degradationCellular protein degradation

highly processive

protein 5-8 a.a.

extremely specificsame cellular compartment, half life can vary from 2 min to many days

“an old hammer is made from two heads and three handles…”

Protein steady state kineticsProtein steady state kinetics

the steady state can be due to high or low k’sthe steady state can be due to high or low k’s

Protein steady state Protein steady state in vivoin vivo

protein

synthesis degradation

to change the concentration of a protein the cell can alter either process

COO-+H3N COO-

COO -

Proteases are well known enzymes…Proteases are well known enzymes…

COO -

+H3N

COO -

+H3N

Proteases for degradationProteases for degradation

classic protease:

highly site specific

not processive

chambered chambered protease:protease:processive and protective

Many chambered proteasesMany chambered proteases

E. coli Rhodococcus Thermoplasma Saccharomyces

versions found in all organismsall function in protein degradation

Eukaryotic 20 proteasomeEukaryotic 20 proteasome

alternative subunits for specialized P’somesalternative subunits for specialized P’somes

0.5 to 1% of the cellular protein0.5 to 1% of the cellular protein

can not degrade folded proteinscan not degrade folded proteins

90o

x

Eukaryotic 20 proteasomeEukaryotic 20 proteasome

28 subunits, 14 separate proteinsactive sites inside; chamber can hold 100kD

how is specificity attained??

Eukaryotic 26S proteasomeEukaryotic 26S proteasome

proteolysissubstrate recognitionsubstrate recognitionATP-dependent unfoldingATP-dependent unfolding

Eukaryotic 26S proteasomeEukaryotic 26S proteasome

QuickTime™ and aPNG decompressor

are needed to see this picture.

Specificity in protein degradationSpecificity in protein degradation

Ub

ubiquitin

76 a.a.8,000 mw

covalent additionof a small tag to markthe targetprotein fordestruction

only in eukaryotesvery highly conserved

Specificity in protein degradationSpecificity in protein degradation

Ub

ubiquitin

76 a.a.8,000 mw

only in eukaryotesvery highly conserved

CO2-Ub

NH3+

Protein

PROTEASOME

UbiquitinationUbiquitination

side chainlysine

UbUb

UbUb

UbUb

Protein

.

.

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

20S core20S coreproteolysisproteolysis19S caps19S caps

binding, unfoldingbinding, unfoldingATP hydrolysisATP hydrolysis

Ubiquitin-proteasome degradationUbiquitin-proteasome degradation

2004 Chemistry Nobel Prize2004 Chemistry Nobel Prize

AronAronCiechanoverCiechanover

AvramAvramHershkoHershko

IrwinIrwinRoseRose

www.nobelprize.orgwww.nobelprize.org

Ubiquitin enzymologyUbiquitin enzymology

ATP

E1- Ub

Ub

E1

ubiquitinUb

E1 ubiquitin activating enzyme

E2E2- UbE2 ubiquitin conjugating enzyme (UBC)

S S-Ub

E3 E3 ubiquitin-protein ligase

S substrate

R IN G -H 2

N

Hrd1p

CxxCx12-35CxHxxHxxCx8-39CxxC

RING-H2

HLSCLKNWMERSQT---CPIC

CTSCLTSWQESEGQG--CPFCHDHCIYRWLDTPTSKGLCPMC

c-CblApc11Hrd1

Ubiquitin ligase (E3) RING domainsUbiquitin ligase (E3) RING domains

M B P

HIS6-huE1

Ub ATP

HIS6-huUBC4

H rd 1 p

R IN G

reaction mix

Δ Ub

each component isrequired

missing item

E1E2 E3 ATP

w.t. C399S

RING-H2dependent

UbGS

T-U

b

multi-Ubaddition

In vitro E3 activity of a RING domainIn vitro E3 activity of a RING domain

The Cell Cycle and UbiquitinThe Cell Cycle and Ubiquitin

L. Hartwell, 2001 Nobel (with Nurse and Hunt)

QuickTime™ and aGIF decompressor

are needed to see this picture.

The Cell Cycle and UbiquitinThe Cell Cycle and Ubiquitin

M-cyclin

S-cyclin

cyclin-dependentkinase

Ub-mediateddegradation

Ub-mediateddegradation

plus at least another layer

The Cell Cycle and UbiquitinThe Cell Cycle and Ubiquitin

Cloning and analysis of CDC genes reveals two main classes of E3 involved in cell cycle control:

1) SCF complex

E3 subunits

2) APC - anaphase promoting complex

over 10 subunits, substrates include B cyclins, Pds1p

F box adaptors for E3 specificityF box adaptors for E3 specificity

F box adaptors for E3 specificityF box adaptors for E3 specificity

CUL1

SKP1

RBX1

F box adaptor

substrate

E2 Ub

RING domain

In many cases, the substrate-F boxinteraction is mediated by phosphorylation

BUT

Oxygen sensing and ubiquitinationOxygen sensing and ubiquitination

CUL1

B/C

RBX1

VHL

HIF1

E2Ub

Cullin1/Rbx1Elongin B/CVHL tum. sup

High O2

Low O2

HIF1

HIF1

Oxygen sensing and ubiquitinationOxygen sensing and ubiquitination

High O2 (HIF degraded)

HIF1 HIF1

OHbinding toCBCVHL E3

Low O2 (HIF stable)

HIF1

prolinehydroxylase

prolinehydroxylase

O2

oxygen-dependentmodification to an E3-binding sub.

An SCF complex in detailAn SCF complex in detail

CUL1

SKP1

RBX1

SKP2

p27

E2 Ub

Coordinates plus docking…Coordinates plus docking…

SCF theme is broadly used in biologySCF theme is broadly used in biology

CUL

SKP1

RBX1

Adaptor

substrate

E2 Ub

Numerous cullins, numerous SKPs, over 50 E2sand a varitey of adaptors:

80 F box200 BTB domain proteins40 SOCS/BT (AYU Box)

Ubiquitination and HIVUbiquitination and HIV

Vpu

CD4

Vpu

HIV-encoded Vpu programs proteasomal degradation of CD4

CD4

VpuF WD40

CulSkp1Rbx

E2

Vpu

HIV-encoded Vpu programs proteasomal degradation of CD4 by recruiting the

cytoplasmic F-box protein TRCP

Ubiquitination and HIVUbiquitination and HIV

p53 E6

E6AP

HECT domains: a distinct E3 familyHECT domains: a distinct E3 family

HPV 16, 18: cancer correlated, p53 degrading

HECT: HECT: HHomology to omology to EE6ap 6ap CC--TTerminuserminus

Homology-driven discovery: p53 againHomology-driven discovery: p53 againMDM2: known regulator of p53 and

transcriptional target

p53MDM2

MDM2 has a functional RING domain

MDM2-p53 regulationMDM2-p53 regulation

MDM2-p53 inhibitorsMDM2-p53 inhibitorsIn Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 (2004) Vassilev et al. Science 303: 844

MDM2-p53 inhibitorsMDM2-p53 inhibitorsIn Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 (2004) Vassilev et al. Science 303: 844

Degradation and immunologyDegradation and immunology

plasma membrane

ER membrane

DRiPs: the source of immune antigens?DRiPs: the source of immune antigens?

DRiPs: the source of immune antigens?DRiPs: the source of immune antigens?

Homology-driven discoveryHomology-driven discovery

Familial Parkinsonism: single gene defect

Model based on E3 function

Responsible gene: Parkin with RING-H2

pathologicalprotein(s)

production Parkinproduction

pathologicalprotein(s)

defectiveParkin

symptoms

Destruction signalsDestruction signals

““degron”degron” distributeddistributedinformationinformation

direct recognitiondirect recognition

rec. of modification (F box)rec. of modification (F box)

regulatoryevents

alteredstructure

Degradation in quality control and Degradation in quality control and protein regulation protein regulation

Quality control in disease Quality control in disease

Alzheimer’sHuntington’sALS (Lou Gehrig’s)Parkinsonismcystic fibrosislong Q-T syndromeretinitis pigmentosaetc...

Alzheimer’sHuntington’sALS (Lou Gehrig’s)Parkinsonismcystic fibrosislong Q-T syndromeretinitis pigmentosaetc...

Ubiquitin in Alzheimer’s plaques Ubiquitin in Alzheimer’s plaques

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

amyloid ubiquitinatedproteins

Quality control degradationQuality control degradation

-operates continuously in all cells

-important part of stress-responsepathways

-particularly important in non-dividing cells such as neurons

-significant therapeutic potential

Quality control ligases of the ERQuality control ligases of the ER

lysines: 6, 11, 27, 29, 33, 48, 63

UbUb

UbUb

UbUb

Alternative linkages for polyubiquitinAlternative linkages for polyubiquitin

Multiple linkage sites in ubiquitinMultiple linkage sites in ubiquitin

Ub

multi-ubiquitin chains

C-48-C-48-C-48

C-29-C-29-C-29

C-63-C-63-C-63

48 linked

29 linked

63 linkedK63-linked chains are not targeted to proteasome

63 linkage in TNF signaling63 linkage in TNF signaling

Ub

63 linkage in TLR signaling63 linkage in TLR signaling

Immune signals

MyD88

IRAK

TRAF6

IKK

Ubc13/Uev1

fraction requiredfor TRAF6 action

TRAF6 catalyzes K63-linkedmulti-ubiquitin chain formation

63 linkage in 63 linkage in TLR signalingTLR signaling

Darwin’s phosphate…Darwin’s phosphate…

altered function

endocytosis of membrane proteins

ribosomal function- L29 protein (2000)

control of vacuolar traffic and delivery (2001)

regulation of DNA repair (2001)

Fanconi’s anemiaFanconi’s anemia

Fanconi’s anemiaFanconi’s anemia

Numerous human complementation groups

A, B, C, D1, D2, E, F, G

Cells senstive to IR and mitomycin C: DNA repair problems

D2 is mono-ubiquitinatedin responseto DNA damagingagents…

Fanconi’s anemiaFanconi’s anemia

D2 is mono-ubiquitinatedin responseto DNA damagingagents…

D2 D2

A, C, E, F, G form a complex required for D2 mono Ub

B = D1 = BRCA2 !!

FANCL is the likely Ub ligase activity (!)

Ubiquitin homologuesUbiquitin homologues

Ubiquitin, SUMO Ubiquitin, SUMO and DNA repairand DNA repair