lys Two strategies for protein degradation 1) Send the protein to a degradative compartment 2) selective degradation of individual molecules

Cellular protein degradation highly processive protein 5-8 a.a. extremely specific same 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 kinetics the steady state can be due to high or low k’s

Protein steady state in vivo protein synthesisdegradation to change the concentration of a protein the cell can alter either process

COO - +H3N+H3N Proteases are well known enzymes… COO - +H3N+H3N +H3N+H3N

Proteases for degradation classic protease: highly site specific not processive chamberedprotease: processive and protective

Many chambered proteases E. coliRhodococcus ThermoplasmaSaccharomyces versions found in all organisms all function in protein degradation

Eukaryotic 20 proteasome alternative subunits for specialized P’somes 0.5 to 1% of the cellular protein can not degrade folded proteins

90 o x Eukaryotic 20 proteasome 28 subunits, 14 separate proteins active sites inside; chamber can hold 100kD how is specificity attained??

Eukaryotic 26S proteasome proteolysis substrate recognition ATP-dependent unfolding

Eukaryotic 26S proteasome

Specificity in protein degradation Ub ubiquitin 76 a.a. 8,000 mw covalent addition of a small tag to mark the target protein for destruction only in eukaryotes very highly conserved

Specificity in protein degradation Ub ubiquitin 76 a.a. 8,000 mw only in eukaryotes very highly conserved

CO 2 - Ub NH 3 + Protein PROTEASOME Ubiquitination side chain lysine Ub Protein

Ub 20S core proteolysis 19S caps binding, unfolding ATP hydrolysis Ubiquitin-proteasome degradation

2004 Chemistry Nobel Prize AronCiechanoverAvramHershkoIrwinRose

Ubiquitin enzymology

Ubiquitin ligase (E3) RING domains

Ub GST-Ub multi-Ub addition In vitro E3 activity of a RING domain

The Cell Cycle and Ubiquitin L. Hartwell, 2001 Nobel (with Nurse and Hunt)

The Cell Cycle and Ubiquitin M-cyclin S-cyclin cyclin- dependent kinase Ub-mediated degradation Ub-mediated degradation plus at least another layer

The 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 specificity

CUL1 SKP1 RBX1 F box adaptor substrate E2 Ub RING domain In many cases, the substrate-F box interaction is mediated by phosphorylation BUT

Oxygen sensing and ubiquitination CUL1 B/C RBX1 VHL HIF1  E2 Ub Cullin1/Rbx1 Elongin B/C VHL tum. sup High O 2 Low O 2 HIF1 

Oxygen sensing and ubiquitination High O 2 (HIF degraded) HIF1  OH binding to CBC VHL E3 Low O 2 (HIF stable) HIF1  proline hydroxylase proline hydroxylase O2O2 oxygen-dependent modification to an E3-binding sub.

An SCF complex in detail CUL1 SKP1 RBX1 SKP2 p27 E2 Ub

Coordinates plus docking…

SCF theme is broadly used in biology CUL SKP1 RBX1 Adaptor substrate E2 Ub Numerous cullins, numerous SKPs, over 50 E2s and a varitey of adaptors: 80 F box 200 BTB domain proteins 40 SOCS/BT (AYU Box)

Ubiquitination and HIV Vpu CD4 Vpu HIV-encoded Vpu programs proteasomal degradation of CD4

CD4 Vpu FWD40 Cul Skp1 Rbx E2 Vpu HIV-encoded Vpu programs proteasomal degradation of CD4 by recruiting the cytoplasmic F-box protein  TRCP Ubiquitination and HIV

p53 E6 E6AP HECT domains: a distinct E3 family HPV 16, 18: cancer correlated, p53 degrading HECT: Homology to E6ap C-Terminus

Homology-driven discovery: p53 again MDM2: known regulator of p53 and transcriptional target p53 MDM2 MDM2 has a functional RING domain

MDM2-p53 regulation

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

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

Degradation and immunology plasma membrane ER membrane

DRiPs: the source of immune antigens?

Homology-driven discovery Familial Parkinsonism: single gene defect Model based on E3 function Responsible gene: Parkin with RING-H2 pathological protein(s) productionParkinproduction pathological protein(s) defective Parkin symptoms

Destruction signals “degron” distributedinformation direct recognition rec. of modification (F box)

regulatory events altered structure Degradation in quality control and protein regulation

Quality control in disease Alzheimer’s Huntington’s ALS (Lou Gehrig’s) Parkinsonism cystic fibrosis long Q-T syndrome retinitis pigmentosa etc... Alzheimer’s Huntington’s ALS (Lou Gehrig’s) Parkinsonism cystic fibrosis long Q-T syndrome retinitis pigmentosa etc...

Ubiquitin in Alzheimer’s plaques amyloid ubiquitinated proteins

Quality control degradation -operates continuously in all cells -important part of stress-response pathways -particularly important in non-dividing cells such as neurons -significant therapeutic potential

Quality control ligases of the ER

lysines: 6, 11, 27, 29, 33, 48, 63 Ub Alternative linkages for polyubiquitin

Multiple 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 linked 29 linked 63 linked K63-linked chains are not targeted to proteasome

63 linkage in TNF signaling Ub

63 linkage in TLR signaling Immune signalsMyD88 IRAK TRAF6 IKK Ubc13/Uev1 fraction required for TRAF6 action TRAF6 catalyzes K63-linked multi-ubiquitin chain formation

63 linkage in TLR signaling

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 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- ubiquitinated in response to DNA damaging agents…

Fanconi’s anemia D2 is mono- ubiquitinated in response to DNA damaging agents… 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 homologues

Ubiquitin, SUMO and DNA repair