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DNA

RNA

PROTEIN

FUNCTION

Protein degradation

Protein synthesisAMINO ACIDS

Some properties of intracellular protein degradation (1970).

• Abnormal proteins are rapidly eliminated.

• Normal proteins are selectively degraded at widely different rates.

• Levels of specific proteins in animal cells can be regulated by

changes in rates of synthesis or rates of degradation (Schimke,

1970).

Hershko & Tomkins JBC 1971

Ciechanover, Hod & Hershko, BBRC 1978

APF-1 (ubiquitin)

Protein (µg) 1.5 3 7 PNAS 1980

ATPlysozyme (µg)

- -+ + + + +0 0 5 10 25 5 5

125I-ubiquitin

125I-lysozyme

lysozyme- (Ub)n

Hershko et al., PNAS 1980

PNAS 1980

DUBDUBDUB

E2E2E2 ProteinProtein

E3E3

UbUb

UbUb

UbUb

UbUb UbUb

UbUb

UbUb

UbUb

UbUb

E2E2E2

E1E1E1

UbUb

E1E1E1

+ATP+ATP

UbUb

AMP+PPAMP+PPiiE1E1E1

E2E2E2

UbUb

++ ATPATP

ADP+PADP+Pii

ProteinProteindegradeddegraded

26S proteasome26S proteasome

UbUb

ProteinProtein

UCHUCHUCH

Hershko et al., JBC 1983

Substrate Yes No Yes No Yes Yes

E3α-125I-proteincross-link

125I-protein

Cytochrome c (yeast)

Cytochrome c (mammalian)

Lysozyme

Ribonuclease A (native)

Ribonuclease A (oxidized 1)

Ribonuclease A (oxidized 2)

DETECTION OF E3-SUBSTRATE BINDING BY CHEMICAL CROSS-LINKING

Free 125I-proteinsJBC 1986

MBC 1995

Cyclosome/APC

MBC 1995

UU

UU

UU

UU

Cdk1

Cyclin B

Cdk1

Cyclin B

Cdk1

~ ~~ ~ Cdc20

PP

InactiveCdk1

InactiveAPC/cyclosome

ActiveAPC/cyclosome

E1, E2-CATP, Ub(b)

ATP

(a)

Cyclinsynthesis

26SProteasome

ActiveCdk1

Phosphatase

(d)

Cyclindegraded

Pi

PP

Cdc20

APC/Cinactive

APC/Cactive

APC/Cinactive

APC/C = Anaphase-PromotingComplex/Cyclosome

G0/G1 TimeTime

Lev

elL

evel

S

p27Cyclin E Cyclin A

Skp2

p27Cyclin E

Cdk2

Cyclin E

Cdk2

Cyclin A

Cdk2

Cdk2 inactive Cdk2 activep27 degraded

Cks1

NCB 1999

NCB 1999

The three binding sites of Cks1

Cdk binding(green)Anion binding

(blue)

Skp2 binding(red)

JBC 2002

p27Cyclin E

Cdk2p27

Cyclin E

Cdk2

Cyclin E

Cdk2

Cdk2 inactive

p27-(Ub)n

P

p27Cyclin E

Cdk2

Skp1

SCF complex

P

Skp2 &Cks1

synthesis

Skp2Cks1

Cyclin Esynthesis

Cdk2 active

p27 degraded

Some roles of ubiquitin-mediated protein degradation.

• Control of cell division• Signal transduction• Regulation of gene expression• Responses to inflammation• Immune response• Embryonic development• Apoptosis• Circadian clocks

Some regulatory proteins degraded by the ubiquitin system.

Regulator Role ofdegradation

Examples

I Positive Limitation of duration

Cyclins (G1, S, M-phase); transcription factors (myc, fos…)

II Negative Initiation of process

Cdk inhibitors Anaphase inhibitorIκB transcriptional regulator

III Positive or negative

Activation by stabilization

p53 tumor suppressor; β-catenin

Type

2000

Microbe hunters

Vitaminhunters

Enzyme hunters

Gene hunters

1900

Arthur Kornberg : For the Love of Enzymes (1989)

Technion lab (1971-present)

Dvora GanothHanna HellerEsther EytanSarah EliasClara SegalJudith Hershko

Former graduate studentsAaron CiechanoverYuval ReissValery SudakinShirly Lahavand many others…

Collaboration and helpIrwin A. RoseLeonard CohenJoan RudermanMichele Pagano

Present graduate studentsGil BornsteinDanielle Sitry-ShevahYakir Moshe