DNA RNA Protein synthesis AMINO ACIDS PROTEIN Protein - PowerPoint PPT Presentation

DNA RNA Protein synthesis AMINO ACIDS PROTEIN Protein degradation FUNCTION

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 ( µ g) 1.5 3 7 PNAS 1980

lysozyme- (Ub) n 125 I-lysozyme 125 I-ubiquitin - - + + + + + ATP lysozyme ( µ g) Hershko et al ., PNAS 1980 0 0 5 10 25 5 5

PNAS 1980

E1 E1 E1 E2 E2 E2 +ATP +ATP E3 E3 Ub E1 E2 Ub E1 E2 E1 E2 E2 E2 E2 Protein Protein Ub Ub AMP+PP i AMP+PP i Ub Ub E1 E1 E1 Ub Ub UCH UCH UCH Ub Ub Ub Ub DUB DUB DUB Ub Ub Protein Protein Ub Ub + + ATP ATP Ub Ub Ub Ub Protein Protein Ub Ub Ub Ub ADP+P i ADP+P degraded degraded i Ub Ub 26S proteasome 26S proteasome

Hershko et al ., JBC 1983

DETECTION OF E3-SUBSTRATE BINDING BY CHEMICAL CROSS-LINKING Ribonuclease A (oxidized 1) Ribonuclease A (oxidized 2) Cytochrome c (mammalian) ) e v i t ) a t s n a ( e A y ( e c s e 125 I-protein e m a m e y l o z c o r u h s n c y o o L b t y i R C Substrate Yes No Yes No Yes Yes E3 α - 125 I-protein cross-link Free 125 I-proteins JBC 1986

MBC 1995

Cyclosome/APC MBC 1995

Inactive APC/cyclosome Cdk1 Cyclin Cyclin B synthesis ATP Active (a) Cdk1 P i Cdk1 (d) Cdk1 Phosphatase Cyclin B E 1 , E 2 - C A T U U P , Inactive U b Cdc20 ( 26S b ) Cdk1 U U Proteasome P P P P U U ~ ~ ~ ~ Active U U ~ ~ ~ ~ Cdc20 APC/cyclosome Cyclin degraded

APC/C APC/C APC/C active inactive inactive APC/C = Anaphase-Promoting Complex/Cyclosome

Cyclin E Cyclin A Level Level p27 Skp2 Cks1 Time Time S G0/G1 Cyclin E Cyclin A Cyclin E p27 Cdk2 Cdk2 Cdk2 Cdk2 inactive p27 degraded Cdk2 active

NCB 1999

NCB 1999

The three binding sites of Cks1 Cdk binding Anion binding (green) (blue) Skp2 binding (red) JBC 2002

SCF complex Skp1 Skp2 & Skp2 Cyclin E Cks1 Cks1 synthesis synthesis P P Cdk2 Cdk2 Cdk2 Cdk2 p27 p27 p27 Cyclin E Cyclin E Cyclin E Cyclin E p27-(Ub) n Cdk2 active Cdk2 inactive 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 of Examples Type degradation Positive Limitation of Cyclins (G1, S, M- I duration phase); transcription factors (myc, fos…) Negative Initiation of Cdk inhibitors II process Anaphase inhibitor I κ B transcriptional regulator Positive or Activation by p53 tumor III suppressor; β -catenin negative stabilization

1900 2000 Microbe hunters Vitamin hunters Enzyme hunters Gene hunters Arthur Kornberg : For the Love of Enzymes (1989)

Technion lab Collaboration and help (1971-present) Irwin A. Rose Leonard Cohen Dvora Ganoth Joan Ruderman Hanna Heller Michele Pagano Esther Eytan Sarah Elias Clara Segal Judith Hershko Present graduate students Former graduate students Gil Bornstein Aaron Ciechanover Danielle Sitry-Shevah Yuval Reiss Yakir Moshe Valery Sudakin Shirly Lahav and many others…