Cellular protein degradationCellular protein degradation

Proteolytic pathways in eukaryotes- lysosomal degradation of proteins- ubiquitin-proteasome dependent protein degradation- post-proteasome degradation: Tricorn, TPII- membrane protein degradation

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Main proteolytic pathways in Main proteolytic pathways in eukaryoteseukaryotes

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

nuclearproteins

cytoplasmicproteins

Mitochondria

Mitochondrialproteolytic

system

ER proteins

endosome-lysosomesystem

cooperation?

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Timeline of regulated intracellular Timeline of regulated intracellular proteolysisproteolysis

adapted from R. John Mayer, Nature reviews 1, 145-148.

19051905 19121912 19421942 19781978 19831983 19841984 19861986 19881988 19911991

ubiquitin structure 1987proteasome structure 1995

19971997

Folin states “endogenousproteins 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

Schoenheimeruses 15N 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

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NobelPrize2004

Aaron CiechanoverAvram Hershko

Irwin Rose

Lysosomal degradation of proteinsLysosomal 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!)

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Lysosomal degradation of proteinsLysosomal 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

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The ubiquitin degradation pathwayThe ubiquitin degradation pathway

E1 - ubiquitin activating enzyme E2 - ubiquitin conjugating enzyme E3 - ubiquitin ligase ‘~’ denotes high-energy thioester bond DUB, deubiquinating enzyme

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Ubiquitin-mediated degradationUbiquitin-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 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

ubO

OHE1-SH

ATPub

OAMP

E1-SH

ubO

S~E1E2

ubO

S~E2

E3prot

ubO

N-H

prot

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E3 ligase

E3 ubiquitin ligasesE3 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-box SCF (Skp1/Cul/F-box)

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E3 ubiquitin ligase: VHL-Elongin B/CE3 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.

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c-CBL E3/E2/kinase structurec-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.

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E2

E3

substrate

E3 E3

E3

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-dependentSCF-dependentubiquitinationubiquitination

in yeastin yeast

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Anaphase promoting complex (APC)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 proteinsconsists 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.

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Tricorn protease of prokaryotesTricorn 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 the730 kDa species

cryo-emreconstructionof tricorn capsids

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void volume! huge

Tricorn protein degradation pathwayTricorn 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 modularsystem forprotein degradation

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Tricorn-like protease in eukaryotes?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 concentrationsof 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

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Membrane protein degradationMembrane 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)

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wt-DHFRstabilisesprotein

degradation

degradation