Presentation is loading. Please wait.

Presentation is loading. Please wait.

Cellular protein degradation Proteolytic pathways in eukaryotes - lysosomal degradation of proteins - ubiquitin-proteasome dependent protein degradation.

Similar presentations

Presentation on theme: "Cellular protein degradation Proteolytic pathways in eukaryotes - lysosomal degradation of proteins - ubiquitin-proteasome dependent protein degradation."— Presentation transcript:

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 17-1

2 Main proteolytic pathways in eukaryotes  endosome-lysosome pathway degrades extracellular and cell- surface proteins  ubiquitin-proteasome pathway degrades proteins from the cytoplasm, nucleus and ER  mitochondria (and chloroplasts) have their own proteolytic system of bacterial origin Nucleus Autophagosome Lysosome/ endosome Ubiquitin- proteasome system nuclear proteins cytoplasmic proteins Mitochondria Mitochondrial proteolytic system ER proteins endosome- lysosome system cooperation? 17-2

3 Timeline of regulated intracellular proteolysis adapted from R. John Mayer, Nature reviews 1, 145-148. 1905 1912 1942 1978 1983 1984 1986 1988 1991 ubiquitin structure1987 proteasome structure1995 1997 Folin states “endogenous proteins are stable” Lewis discovers ‘bodies’ in patients with Parkinson’s disease Hershko and Ciechanover discover the process of ubiquitylation Varshavsky, Ciechanover and Finley discover ubiquitylation is essential for viability and cell-cycle progression Schoenheimer uses 15 N to show continuous protein turnover Hershko et al. identify enzymes of the ubiquitin- protein ligase system 26S proteasome partly purified by Rechsteiner Kischner et al. discover that cyclin is degraded by the ubiquitin pathway Lowe, Landon and Mayer discover that Lewy bodies are full of ubiquitylated proteins Several groups discover the combinatorial control and specificity of SCF ubiquitin protein ligases 17-3 Nobel Prize 2004 Aaron Ciechanover Avram Hershko Irwin Rose

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!) 17-4

5 Lysosomal degradation of proteins Cuervo and Dice (1998) J. Mol. Med. 76, 6-12.  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 17-5

6 The ubiquitin degradation pathway  E1 - ubiquitin activating enzyme  E2 - ubiquitin conjugating enzyme  E3 - ubiquitin ligase  ‘~’ denotes high-energy thioester bond  DUB, deubiquinating enzyme 17-6

7 Ubiquitin-mediated degradation  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 NH 2 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 ub O OH E1-SH ATP ub O AMP E1-SH ub O S~E1 E2 ub O S~E2 E3 prot ub O N-H prot 17-7

8 17-7b E3 ligase

9 E3 ubiquitin ligases  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 ubiquitin- like protein rub-1; ElonginB also has homology with ubiquitin  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) SH2, WD40, Ank, LRR are all protein-protein interaction domains VHL/SOCS-boxSCF (Skp1/Cul/F-box) 17-8

10 E3 ubiquitin ligase: VHL-Elongin B/C  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. 17-9

11 c-CBL E3/E2/kinase structure  c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases (e.g., ZAP-70), promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates 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. 17-10 E2 E3 substrate E3

12  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 SCF-dependent ubiquitination in yeast 17-11

13 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 unprocessed em images em reconstruction Gieffers et al. (2001) Mol. Cell 7, 907-913. 17-12

14 Tricorn protease of prokaryotes Walz et al. (1997) Mol. Cell 1, 59-65.  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 (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 cryo-em reconstruction of tricorn capsids 17-13 void volume! huge

15 Tricorn protein degradation pathway Yao and Cohen (1999) Curr. Biol. 9, 551-553.  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  probably one of many pathways of protein degradation in prokaryotes a modular system for protein degradation 17-14

16 Tricorn-like protease in eukaryotes? Geier et al. (1999) Science 283, 978-981.  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- 4- methylcoumarin (AAF-AMC)-hydrolyzing activity in size exclusion fractions larger than proteasome  forms on average tripeptides  general proteolytic activity Legend to gels: Galactosidase (116 kD) (lane 1), purified TPPII (lane 2), fast- and slow- running electrophoretic isoforms of 26S proteasomes (lanes 3 and 4, respectively), and purified 20S proteasomes (lane 5) em pictures of TPPII; dumbbell- or ovoid-shaped 17-15

17 Membrane protein degradation  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. p=precursor; m=mature; 25ºC=no unfolding; 37ºC=unfolding of domain(s) 17-16 wt-DHFR stabilises protein degradation

Download ppt "Cellular protein degradation Proteolytic pathways in eukaryotes - lysosomal degradation of proteins - ubiquitin-proteasome dependent protein degradation."

Similar presentations

Ads by Google