Topological Diversity of Proteolytic Systems 1. Extracorporeal Extracellular - The gastrointestinal tract

2. Intracorporeal Extracellular - The blood coagulation system

3. Intracorporeal Intracellular - The Lysosome (organelle - membrane secluded) and the Ubiquitin system (free floating- cytosolic, nuclear, “ER”) Intracellular Proteolysis is a thermodynamically paradoxical, energy (ATP)-requiring process, Schoenheimer, R. (1942). The Dynamic State of Body Constituents. Harvard University Press, Cambridge, USA.

“The simile of the combustion engine pictured the steady flow of fuel into a fixed system, and the conversion of this fuel into waste products. The new results imply that not only the fuel, but the structural materials are in a steady state of flux. The classical picture must thus be replaced by one which takes account of the dynamic state of body structure”.

The Lysosome

Courtesy: Viola Oorschot and Judith Klumperman, Department of Cell Biology, University of Utrecht School of Medicine, Utrecht, The Netherlands Ciechanover, 2005

Ciechanover et al., 1978 Ciechanover et al., 1978 Ciechanover et al., 1980 Ubiquitin Generates Covalent Adducts With Proteolytic Substrates 123456 7

C6 C5 C4

C3 C2? Cont.

C1?

Hershko et al., 1980 Hershko et al., 1980 E3

E3 E2 UB E1 UB UB

P Target UB UB UB E3 P UB UB P Target UB UB UB P UB UB P Target UBUB UBUB UB P UBUB UBUB

Hershko et al., 1983

Ciechanover et al., 1984

Ciechanover et al., 1985 Conversion of acidic N-terminus into a basic residue is required for E3α recognition

1.

Basic N-terminal residue E3α (Arg, His, Lys) B Ubiquitination and degradation

2. Arg tRNA-protein Acidic N-terminal residue transferase E3α (Asp, Glu) A R A Arg-tRNA

Ubiquitination and RNase degradation Ciechanover et al., 1988 Ciechanover et al., 1991 Gropper et al., 1991 Ciechanover et al., 1997 Bercovich et al., 1997 Gonen et al., 1996 Breitschopf et al., 1998 The ubiquitin system, its allies, and some of their many functions

Ciechanover, 2005 Protein Substrate Ancillary or Protein viral protein The Ubiquitin Proteasome Pathway Substrate

E Accelerated 3 Ub E+ E E + 1 2 3 degradation Protein Substrate Normal degradation rates Ub Ub Ub maintain cellular proteins in an appropriate, Ub Ub Ub though dynamic, Peptides steady state x 26S Protein Decreased Substrate degradation Protein Substrate x

Mutation in a system enzyme or in a substrate’s recognition motif AberrantAberrant ProteinProtein DegradationDegradation LeadsLeads toto DiseaseDisease

Oncogenic Tumor Suppressor Proteins Proteasome Proteins

Cancer Cancer

Decreased Normal Increased Degradation Degradation Degradation

Structure of the Yeast Proteasome and CP

Proteasome CP Top view of CP Inhibition of NF-kB Stabilization of cell-cycle regulatory proteins (p27, p53) Induction of apoptosis Weak MDR substrate Overcomes Bcl-2 cytoprotection CompleteComplete ResponseResponse toto PSPS--341341

• 3 prior transplants, Ch 13 abnormal • MP, VAD, Mel/PBSC x 3, dexamethasone • Gemzar, DCEP, dendritic vaccine • Thalidomide, DCEAP, Doxil/Navelbine 2.5 PS-341 2.0 IgA 1.5 g/dL 1.0 0.5 CR 0.0 10/1/00 12/1/00 2/1/01 4/1/01 6/1/01 8/1/01 10/1/01 BM 50% <5% PC% Patient 004 : Marrow Biopsies

• Pre-PS-341: 41% plasma • Post-PS-341: 1% cells Patient 010: Refractory Follicular NHL 2.5 x 1.2 cm 0.4 x 0.4 cm

• Pre-treatment • After cycle 1 of PS-341 Nutlin-2 Binds to the p53 pocket of MDM2 (side chains of Phe 19, Trp 23 and Leu 26)

Vassilev et al., Science 2004 Effect of Nutlin-3 (P.O. ) and Doxorubicin (I.V.) on SJSA-1 human cancer xenograft in nude mice

Vassilev et al., Science 2004 Mentors, Research Associates, Graduate Students, Post-doctoral Fellows, Collaborators and Guest Scientists Graduate Students: Mentors: Sara Ferber Collaborators: Ronit Gropper Ya’acov Bar Tana Alexander Varshavsky Arie Mayer Benjamin Shapira Daniel Finley Joseph DiGiuseppe Avram Hershko Alan Schwartz Hedva Gonen Ernie Rose Shlomit Gross-Mesilaty Kazuhiro Iwai Dganit Burd Harvey Lodish Jacob I. Sznajder Nava Bluemenfeld Dalia Dickman Research Associates: Post-doctoral Fellows: Ossama Abu Hatoum Sarah Elias Ilana Stancovski Assaf Meytav Hedva Gonen Amir Oryan Kristin Breitschopf Aviad Hoffman Beatrice Bercovich Siegal Aviel Eyal Reinstein Ruth E. Amir Ifat Fajerman Guest Scientists: Cynthia Guimaraes Hillit Achbert Stuart Arfin Limor Dori Shirly Baratz-Lahav Sammer Diab (at A. Hershko’s Laboratory) Ifat Fajerman Ronen Ben-Saadon Yair Argon Yifaat Hermann-Bachinsky Tahel Kaufman Liron Torres Dafna Zaarur