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)