Transcript lecture 17
17-1 Cellular protein degradation Proteolytic pathways in eukaryotes - lysosomal degradation of proteins - ubiquitin-proteasome dependent protein degradation - post-proteasome degradation: Tricorn, TPII - membrane protein degradation Main proteolytic pathways in eukaryotes Mitochondrial proteolytic system endosomelysosome system Mitochondria cytoplasmic proteins Ubiquitinproteasome system Lysosome/ endosome Autophagosome ER proteins nuclear proteins Nucleus 17-2 endosome-lysosome pathway degrades extracellular and cellsurface proteins ubiquitin-proteasome pathway degrades proteins from the cytoplasm, nucleus and ER mitochondria (and chloroplasts) have their own proteolytic system of bacterial origin 17-3 Timeline of regulated intracellular proteolysis Schoenheimer uses 15N to show continuous protein turnover Folin states “endogenous proteins are stable” 1905 1912 Lewis discovers ‘bodies’ in patients with Parkinson’s disease 1942 1978 Hershko and Ciechanover discover the process of ubiquitylation ubiquitin structure 1987 proteasome structure 1995 Hershko et al. identify enzymes of the ubiquitinprotein ligase system 1983 1984 26S proteasome partly purified by Rechsteiner 1986 Varshavsky, Ciechanover and Finley discover ubiquitylation is essential for viability and cell-cycle progression 1988 Kischner et al. discover that cyclin is degraded by the ubiquitin pathway 1991 Lowe, Landon and Mayer discover that Lewy bodies are full of ubiquitylated proteins Aaron Ciechanover Avram Hershko Irwin Rose 1997 Several groups discover the combinatorial control and specificity of SCF ubiquitin protein ligases adapted from R. John Mayer, Nature reviews 1, 145-148. Nobel Prize 2004 17-4 Lysosomal degradation of proteins lysosomes are cellular vesicles containing proteolytic enzymes (e.g., papain-like cysteine protease, serine proteases, aspartic proteinases, etc., which are typically monomeric pH maintained at ~5.5 by proton-pumping ATPase account for 1-15% of cell volume (most abundant in liver and kidney) Most lysosomal enzymes are transported to lysosomes through recognition by receptors for mannose-6-phosphate. Lysosomal enzymes are synthesized like proteins destined to be secreted or for residence on the plasma membrane but are recognized by a phosphotransferase enzyme shortly after leaving the ER. This enzyme transfers N-acetylglucosamine-1-phosphate to one of more mannose residues. A glucosaminidase next removes the glucosamine to generate the M6P. a mutation in the transferase leads to disease (I-cell disease); other so-called lysosomal storage diseases are the Tay-Sachs syndrome (ganglioside accumulates due to beta-Hexosaminidase deficiency), Pompe disease (accumulation of glycogen due to lack of -Glucosidase), etc. (6 others!) Lysosomal degradation of proteins 17-5 macroautophagy is the equivalent of forming intracellular endosomes (phagosomes) that fuse to the lysosome and result in the breakdown of its contents Hsc73 (constitutively-expressed Hsp70 chaperone) is involved in one pathway of lysosome-mediated degradation Cuervo and Dice (1998) J. Mol. Med. 76, 6-12. 17-6 The ubiquitin degradation pathway E1 - ubiquitin activating enzyme E2 - ubiquitin conjugating enzyme E3 - ubiquitin ligase ‘~’ denotes high-energy thioester bond DUB, deubiquinating enzyme Ubiquitin-mediated degradation O ub O E1-SH OH ATP ub AMP E1-SH O ub O N-H ub prot prot S~E1 E2 E3 O ub S~E2 17-7 E1 - ubiquitin activating enzyme uses ATP to activate the carboxyl group of ubiquitin’s C-terminal residue (Gly76). The outcome of this reaction is the formation of a thioester between Gly76 of ubiquitin, and a cysteine residue of E1 E2 - ubiquitin conjugating enzyme accepts the ubiquitin from the E1 through a thioester linkage with a cysteine E3 - ubiquitin ligase transfers the ubiquitin molecule to the epsilon NH2 group of lysine on the substrate ubiquitin molecules are then added in succession to the Lysine 48 residue to form a multiubiquitin chain the DUB enzyme ‘recycles’ ubiquitin the 26S proteasome degrades the substrate to peptides 17-7b E3 ligase 17-8 E3 ubiquitin ligases there are two basic types of E3 ubiquitin ligases: those possessing Ring fingers (e.g., VHL, SCF, APC, MDM2, c-CBL, etc.) those possessing HECT domains (E6AP-related proteins) Shown here are VHL and SCF ubiquitin ligases. They both associate with Rbx-1, an evolutionarily conserved protein containing a so-called ‘ring finger’ (not shown in figure) ring fingers are also present in other ligases such as the APC and MDM2, which is involved in ubiquitinating p53 CDC34 is modified with the ubiquitinlike protein rub-1; ElonginB also has homology with ubiquitin SH2, WD40, Ank, LRR are all protein-protein interaction domains VHL/SOCS-box SCF (Skp1/Cul/F-box) E3 ubiquitin ligase: VHL-Elongin B/C 17-9 the domain serves to target proteins for degradation; HIF (hypoxia inducible factor) is one of the targets VHL mutations cause tumours (VHL surface mapped with common mutations): crystal structure of a core ubiquitin ligase Stebbins et al. (1999) Science 284, 455-461. 17-10 c-CBL E3/E2/kinase structure c-Cbl proto-oncogene is a RING family E3 that substrate recognizes activated receptor tyrosine kinases (e.g., ZAP-70), promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates E3 signaling crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families Zheng et al. (2000) Cell 102, 533-539. E3 E3 E2 E3 17-11 SCF-dependent ubiquitination in yeast F-box proteins mediate substrate selectivity in degrading various yeast proteins many (all?) of the substrates need to be phosphorylated to be recognized by the F-box protein WD40 and leucine-rich repeats (LRRs) present in F-box proteins mediate protein-protein interactions 17-12 Anaphase promoting complex (APC) The anaphase-promoting complex (also termed ‘cyclosome’) is a ubiquitin-protein ligase that controls important transitions in mitosis by ubiquitinating regulatory proteins consists of many different proteins, including some related to SCF (e.g., ring protein) To initiate sister chromatid separation, the APC has to ubiquitinate the anaphase inhibitor securin, whereas exit from mitosis requires the ubiquitination of B-type cyclins em reconstruction unprocessed em images Gieffers et al. (2001) Mol. Cell 7, 907-913. 17-13 Tricorn protease of prokaryotes tricorn protease is a huge hexameric protease complex that assembles into even larger cage-like structure containing 20 hexamers (14.6 MDa) cage required for efficient degradation? example of self-compartmentalization void volume! huge cryo-em reconstruction of tricorn capsids (A) tricorn protease exists as 2 different species; one of ~730 kDa and one much larger which elutes in the void volume of the sizing column (B) electron microscopy (em) of the 730 kDa species Tricorn protein degradation pathway 17-14 tricorn protease in prokaryotes may be part of a degradation pathway that involves proteasome (in archaea) or other ATP-dependent proteases in archaea/bacteria proteasomes/other oligomeric proteases digest proteins to small peptides tricorn protease then cleaves these to 2-4 mers, which are then degraded down to the level of free amino acids by aminopeptidases a modular system for protein degradation probably one of many pathways of protein degradation in prokaryotes Yao and Cohen (1999) Curr. Biol. 9, 551-553. Tricorn-like protease in eukaryotes? tripeptidyl peptidase II (TPPII) is a cytosolic subtilisin-like peptidase that may be functionally related to Tricorn protease discovery cells adapted to near-lethal concentrations of vinyl sulphone (VS)-proteasome inhibitors still have the ability to degrade ubiquitinated proteins, control the cell cycle, and present MHC class I peptides cells had alanyl-alanyl-phenylalanyl-7-amino- 4methylcoumarin (AAF-AMC)-hydrolyzing activity in size exclusion fractions larger than proteasome forms on average tripeptides em pictures of TPPII; dumbbell- or ovoid-shaped general proteolytic activity Legend to gels: Galactosidase (116 kD) (lane 1), purified TPPII (lane 2), fast- and slowrunning electrophoretic isoforms of 26S proteasomes (lanes 3 and 4, respectively), and purified 20S proteasomes (lane 5) Geier et al. (1999) Science 283, 978-981. 17-15 Membrane protein degradation 17-16 AAA proteases mediate the degradation of membrane proteins in bacteria, mitochondria and chloroplasts (i.e., compartments of eubacterial origin) bacterial Lon, FtsH combine proteolytic and chaperone activities in one system, acting as quality-control machineries - model substrate polypeptides containing hydrophilic domains at either side of the membrane can be completely degraded by either of two AAA proteases found in mitochondria, if solvent-exposed domains are in an unfolded state - a short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side Leonhard et al. (2000) Mol. Cell 5, 629-638. wt-DHFR stabilises protein degradation degradation p=precursor; m=mature; 25ºC=no unfolding; 37ºC=unfolding of domain(s)