23-1
Quality Control
“QC”
- folding or degradation?
- Hsp90, CHIP, UFD2
23-2
Quality control: folding or degradation?
refolding
non-native
protein
refolding
Native
protein
Native
protein
unfolding
degradation
peptides,
amino
acids
23-3
QC
 Cells must ensure a proper Quality Control mechanism over all proteins in
the cell, throughout their lifetimes
 Quality control normally involves:
 proper biogenesis of proteins; maintenance of
folded/assembled/functional conformation; proper cellular localization
 degradation of proteins when required
 A protein triage mechanism, mostly performed by chaperones and
proteolytic degradation machineries, exists
 during normal and in particular during stress conditions
 for soluble and membrane-bound proteins (Lon, FtsH, etc.)
 ERAD (ER-Associated Degradation) represents a quality control
mechanism that operates in conjunction with the chaperones involved in
glycoprotein biogenesis
 AAA ATPases are well suited for quality control, but numerous other
chaperones/chaperone cofactors are involved (e.g., BAG-1)
 the proteasome, lysozome pathways are the predominant machineries
required for protein degradation
23-4
Hsp90 in protein triage
 Hsp90 cooperates with numerous cofactors
(Hsp70, HIP, HOP, p23, cyclophilins) to assist
the maturation/activation of kinases, transcription
factors, etc.
 Hsp90 forms a complex with unstable
firefly luciferase
 there is also evidence that Hsp90 plays a role
in quality control
(A) determination of firefly luciferase activity after
a 10-minute heat shock in the presence or absence of
Herbimycin A (HA), a specific Hsp90 inhibitor
(B) quantitation of 35S-labeled luciferase after heat
shock in the presence or absence of HA
Results show that Hsp90 is implicated
in the folding/degradation of luciferase
(and other ‘typical’ substrates, e.g. kinases)
yeast cells
HSP90dependent
folding
recovery of
activity
- HA
no recovery
+ HA
HSP90-dependent degradation
- HA
+ HA
Schneider et al. (1996) Pharmacologic shifting of a balance
between protein refolding and degradation mediated by
Hsp90. Proc. Natl. Acad. Sci. USA 93, 14536-41.
23-5
CHIP: a novel co-chaperone involved
in quality control
CHIP:
Carboxy terminus of Hsp70-Interacting Protein
 CHIP, a 35 kDa protein, was previously identified as a protein that binds Hsp70
 immunoprecipitates of Hsp70 contain Hsp40, Hsp90, HIP, HOP, BAG, as well
as CHIP and other proteins
 as with Hsp70 cofactors, CHIP modulates the ATPase activity of Hsp70
 CHIP inhibits the ATP-stimulating activity of Hsp40 [opposite of BAG-1]
 domain structure of CHIP:
TPR repeats charged region
U-box
 the U-box represents a modified form of the ring-finger motif that is found in
ubiquitin ligases and defines the E4 family of polyubiquitination factors (UFD2)
Connell et al. (2000) The co-chaperone CHIP regulates protein triage decisions mediated by
heat-shock proteins. Nat. Cell Biol. 3, 93-96.
Meacham et al. (2000) The Hsc70 co-chaperone CHIP targets immature CFTR for
proteasomal degradation. Nat. Cell Biol. 3, 100-105.
23-6
Function of CHIP
start
both Bag and CHIP interact with Hsp70 and
have proteasome-targeting domains
assist
folding
assist
degradation
Modulation of the Hsp70 chaperone cycle by Bag-1 and CHIP. Hsp70 (dark blue, ATPase domain; light blue, substrate-binding domain)
interacts with non-native substrates in a low-affinity ATP conformation (substrate binding domain open) or a high-affinity ADP conformation
(substrate binding domain closed). Substrates are locked in the ADP conformation, and thereby shielded from aggregation, by rapid, Hsp40stimulated ATP hydrolysis. Subsequent nucleotide exchange recycles Hsp70 to the ATP state and leads to substrate release, enabling
substrates to fold to their native conformation [2]. At low concentrations, free Bag-1 accelerates nucleotide exchange via its BAG domain in
a manner productive for substrate folding [10] (right cycle). In contrast, nucleotide exchange and substrate release stimulated by Bag-1
bound to the 26 S proteasome via its UBL domain is proposed to mediate efficient substrate degradation [5,17] (left cycle). For simplicity,
substrate ubiquitination is not shown. The mechanism of negative regulation by CHIP is not known in detail. CHIP binds to the carboxyterminal region of Hsp70 via its TPR domain and inhibits Hsp40-stimulated ATP hydrolysis [11], thereby probably interfering with tight
substrate binding. Bag-1 and CHIP domains are colour-coded according to Fig. 1.
Wiederkehr et al. (2002) Protein Turnover: A CHIP Programmed for Proteolysis. Curr. Biol. 12, R26-28.
Function of CHIP
assists
folding
assists
degradation
Re-modelling of chaperone–glucocorticoid
receptor (GR) complexes by CHIP. Ordered,
nucleotide-dependent interactions of Hsp70,
Hsp90 and the co-chaperones Hop and p23
with folding competent GR molecules are
necessary for hormone (H)-induced folding of
GR (top; reviewed in [20]). Alternatively,
CHIP binding via its TPR domains to Hsp70
and/or Hsp90 induces dissociation of p23 and
Hop from the chaperone–GR complex.
Specific ubiquitin conjugating enzymes (E2s)
are recruited to the U-box of CHIP and
catalyze the attachment of ubiquitin (Ub)
chains to GR (bottom).
23-7
23-8
UFD2:
a novel family of
ubiquitin ligases
Description
 the UFD2 family of proteins are highly
conserved and have a U-box (modified ring
finger as the common motif)
 CHIP is the only member that has a
TPR domain
 ARM domain is an ATP-Regulated
Module found in numerous proteins
Functions
 required for the multiubiquitination of
proteins following E1-E2-E3 ‘activation’ of
substrates
 UFD2-related proteins in plants are
involved in development, and yeast UFD2 is
linked to cell survival under stress conditions
Discovery of UFD2
Johnson et al. (1995) A proteolytic
pathway that recognizes ubiquitin as a
degradation signal. J. Biol. Chem. 270,
17442-56. (Varshavsky lab)
After characterization of genes
involved in the ubiquitin pathway,
the authors found that:
“UFD2 and UFD4 appear to influence
the formation and topology of a multi-Ub
chain linked to the fusion's Ub moiety”
Koegl et al. (1999) A novel
ubiquitination factor, E4, is involved in
multiubiquitin chain assembly. Cell 96,
635–644. (Jentsch lab)
After purification of a protein that
interacted with a ubiquitinated GSTubiquitin fusion protein:
“In fact, UFD2 had been discovered
previously in a genetic screen for mutants
that stabilize UFD substrates (Johnson et
al., 1995 ). Its function in the proteolytic
pathway, however, has remained unclear”
1, Ubi-GST + yeast extract
>>> eluted proteins
2, ubiquitinated Ubi-GST + extract >>> eluted proteins
Koegl et al. showed that E1, E2, E3, E4 can mediate the
multiubiquitination of a sustrate in vitro; E4 functions as a
ubiquitin-chain assembly factor.
E4 associates with CDC48, a AAA ATPase whose
homologue (p97) is known to bind at least one type of
ubiquitinated protein
23-9
24-1
Protein degradation diseases
Degradation and disease
- aggresomes and russell bodies: cellular indigestion
- neurodegeneration and polyglutamine aggregates
- others
24-2
Quality control in the ER
membranebound
chaperone
newly-imported
ER protein
is quickly
glycosylated
PDI
glycotransferase
folding sensor
soluble
lectin/
chaperone
protein concentration in ER is extremely high
PDI
24-3
Degradation of abnormal ER proteins
ERAD
 Proteins that fail to fold
properly in the ER are
normally degraded by
chaperone-mediated
targeting out of the
organelle, ubiquitinated,
and degraded by the
proteasome
 protein misfolding is
typically caused by
mutations or inefficient
biogenesis of particular
proteins (e.g. CFTR)
 what if a protein cannot be degraded?
Aggresomes and Russell bodies
e.g. IgG chain
24-4
 abnormal proteins need to be
disposed of, or else they end up
in ‘inclusions’:
 ER
 Russell bodies
 aggresomes
 proteins that are cytosolic
can also end up in aggresomes
 process of aggresome
formation depends on
microtubules (MTs) and MTbased motor (dynein)
Dislocation and degradation are critical steps for the disposal of misfolded proteins in the ER. Failure of the former may perturb
homeostasis, leading to the accumulation and aggregation of proteins in the ER lumen. Aggregates, which may be ordered or not,
are often sorted into Russell bodies—subregions of the rough ER that tend to exclude soluble chaperones and other normal proteins
present in the ER lumen. Failure of the proteasome to degrade dislocated proteins leads to the accumulation of polyubiquitylated,
deglycosylated proteins in the cytosol. Aggregates are sequestered in aggresomes by retrograde transport on microtubules (gray
track), facilitated by cytoplasmic dynein (red)–dynactin (green) complexes.
Cellular indigestion
black arrow=ribosome on RB
white=normal ER
If the synthesis rate for any given protein
exceeds the combined rates of folding and
degradation, some of the protein will
accumulate in a misfolded/aggregated form.
Russell bodies
- Russell bodies arise from ER-derived
aggregated proteins (e.g., mutant Ig chains)
- Aggresomes arise from misfolded protein
aggregates in the cytosol. They are formed
around the microtubule organising centre,
and contain, in addition to the misfolded
protein, proteasome subunits and
chaperones.
Aggresomes
Inclusion bodies
- Inclusion bodies are bacterial cytosolic
structures that contain misfolded/aggregated
protein, as well as IbpA and IbpB (small Hsp
molecular chaperones)
24-5
24-6
Proteins that form aggregated cellular
inclusions
 ER proteins
 CFTR. delta-508 mutation is the most common cause of Cystic Fibrosis,
and makes biogenesis of membrane protein even less efficient
 Immunoglobulins. Somatic hypermutation of Ig, especially visible in
plasma cells
 alpha1 anti-trypsin. Accumulation causes deposits in hepatocytes,
resulting in liver disease
 Proteins involved in neural processes
 neurodegeneration: alpha-synuclein (Parkinson’s), Alzheimer’s disease,
huntington’s disease, prion disease, etc.
 Bacterial proteins
 inclusion bodies
process of aggregation
is the cause of
cytotoxicity?
abnormal
protein
aggregates themselves
is the cause of
cytotoxicity?
aggregates
objective
set up a system where one can monitor
the in vivo level of proteasome activity in a mammalian
model for a misfolding disease
Molecular mechanism of Disease
24-7
effect of impairing the proteasome system with a protein that forms aggresomes
GFP
GFPu
proteasome
inhibitors
protease
inhibitors
 GFPu is GFP fused to a
short ‘degron’, or
degradation signal at the Nterminus
 cells expressing GFPu
were designated GFPu-1
 DMSO is the mocktreated cells (the protesome
inhibitors are all disolved in
DMSO)
 result: GFP is a degraded
by the ubiquitinproteasome system
GFPu is a substrate of the ubiquitin-proteasome system. (A) Pulse-chase analysis of GFP and GFPu. (Left) Fluorograms of anti-GFP
immunoprecipitates sampled at the indicated chase times in the presence or absence of lactacystin. (Right) Quantification of pulse-chase
data for GFPu (squares) and GFP (circles) in the presence (closed symbols) or absence (open symbols) of lactacystin. (B) Steady-state
level of GFPu after 5-hour treatment of GFPu-1 cells with the indicated protease inhibitors. (C) Lysates of untransfected HEK or GFPu1 cell were treated overnight with the proteasome inhibitor ALLN, or mock-treated, as indicated, immunoprecipitated with anti-GFP, and
immunoblotted with a ubiquitin monoclonal antibody.
Bence et al. (2001) Science 292, 1552-5.
24-8
+ALLN
GFPu
GFP
flow cytometry
+ proteasome
inhibitor
- inhibitor
biochemical assay
of proteasome
(chymotrypsin-like activity)
GFPu
inhibition of
proteasome act.
GFPu fluorescence is a sensitive measure of
UPS (ubiquitin-proteasome system) activity
in vivo. (A) GFPu-1 cells before (left) and after
(right) incubation with lactacystin (6 µM). (B)
Time course of fluorescence in the presence of
ALLN (10 µg/ml), assessed by flow cytometry.
GFPu-1 cells (black circle ), HEK cells (white
circle ), and GFP-expressing cells (white
square). (C) Degradation kinetics of GFPu.
Fluorescence of GFPu-1 cells (squares) or
stable GFP-expressing cells (circles), assessed
by flow cytometry. After a 3-hour incubation
with ALLN, cells were incubated with emetine
in the presence (closed symbols) or absence
(open symbols) of ALLN (10 µg/ml). (D) GFPu
fluorescence is a dynamic indicator of UPS
activity. GFPu-1 cells were incubated with
lactacystin. Relative GFPu fluorescence (black
square ), assessed by flow cytometry, and
relative inhibition of chymotrypsin-like
proteasome activity (black circle ), determined
from lysates of lactacystin-treated cells. (E)
The percentage proteasome inhibition from (D)
plotted against GFPu fluorescence.
% proteasome
inhibition relative to fluorescence
 result: GFPu can be used as a reported of the UPS activity in vivo, especially under
conditions where the UPS is inhibited
cells expressing Flag-F508 CFTR
24-9
aggregates
only cell with
aggregate has GFPu
fluorescence
Protein aggregates inhibit the UPS. (A) GFPu-1 cells
transiently transfected with FLAG- F508 imaged for FLAG
immunofluorescence or GFPu fluorescence. The arrow
indicates a cell containing a FLAG- F508 aggresome. (B).
Quantitative analysis of data in (A) showing GFPu
fluorescence (ordinate) in a subpopulation of FLAG- F508transfected GFPu-1 cells exhibiting high (top 3%) FLAGF508 expression compared with GFPu fluorescence in the
subpopulation containing lower (middle 50%) FLAG- F508
expression. (C) GFPu fluorescence, in FLAG- F508transfected GFPu-1 cells with (bottom) or without (top)
FLAG-immunoreactive aggresomes. (D) GFPu-1 cells
transiently transfected with Q25-MYC or Q103-MYC
imaged for huntingtin expression (MYC
immunocytochemistry) or GFPu fluorescence (bottom).
Inclusion bodies are present in some huntingtin-expressing
cells (arrows), but not in others (arrowheads). (E)
Quantification of data from (D). GFPu fluorescence in GFPu1 cells expressing Q25-MYC (top) or Q103-MYC (bottom)
with inclusion bodies larger than 400 pixels. (F) Correlation
between GFPu fluorescence and inclusion area in Q103MYC-transfected GFPu-1 cells.
 result: link between protein
aggregation and inhibition of UPS
24-10
lo: low aggregation
hi: high aggregation
(propidium iodide)
Protein aggregation induces accumulation of ubiquitin conjugates and cell cycle arrest. (A) Ubiquitin immunoblot of lysates of HEK cells
transfected with either Q25-GFP or Q103-GFP, as indicated, and sorted into populations containing the lowest or highest 10% of GFP
fluorescence. Each lane contains lysates from ~40,000 cells. (B) Two-parameter FACS profiles of HEK cells transfected with GFP, Q25GFP, or Q103-GFP. GFP fluorescence is plotted against DNA content (propidium iodide fluorescence). The fluorescence signals in the two
channels are indicated by pseudocolor, with "hot" colors (i.e., red) being highest and "cold" colors (i.e., blue) lowest. TO INTERPRET
WITHOUT THE USE OF COLOUR: the RED HOT-SPOT in panel 1 of (B) is localized in the lower-left corner, under the 2n; the hotspot in the middle panel of (B) is spread out a bit more, but is still under the 2n; the red hot-spot of the third panel in (B) is on the upper
right-hand side, above the 4n.
Interpretation of results: cells defective in ubiquitin conjugation or exposed to proteasome
inhibitor arrest primarily at the G2/M boundary of the cell cycle. To assess the effect of protein
aggregation on the cell cycle, we transfected HEK 293 cells with GFP, Q25-GFP, or Q103-GFP and
analyzed the cells by flow cytometry for GFP fluorescence and DNA content (Fig. 4B). Cells with
the highest level of expression of Q103-GFP had 4n DNA content, indicating arrest in G2. No such
subpopulation of cells was observed in cells expressing comparable levels of Q25-GFP or GFP (Fig.
4B).
 result: protein aggregation causes cell-cycle arrest
Disease prevention:
ataxin-1 as an example
Ataxin-1 Human ataxin-1 is encoded by the gene Spinocerebellar ataxia type 1
(SCA1), which results in a neurodegenerative disease if it is modified by an
expansion in a polyglutamine tract
Question: what proteins can modify
the toxicity of a protein that
aggregates in vivo?
Approach: express wild-type, 30Q
and 82Q forms of the protein in the
Drosophila eye and carry out a
genetic screen to identify genes that
alter the degenerative phenotype
24-11
Ataxin-1 in Drosophila: the phenotype
24-12
linked to:
Spinocerebellar ataxia
UAS, Upstream Activating Sequence
(for expression in Drosophila eye)
Polyglutamine (CAG)
repeats
using strain
harbouring the
GMR-GAL4
 Strong ataxin-1 eye phenotypes are
produced by the 82Q construct
 see abnormal eye morphology (a-c),
and retinal degeneration (d-f)
 Weaker ataxin-1 phenotypes are observed
with the 30Q construct
 surprising: expect nothing, but
expression is very strong
 higher temperatures increase the severity
of the phenotype
control
30Q
80Q
 overexpression of 82Q and 30Q cause
similar phenotypes in mice cerebellum
(neurodegeneration)
Ataxin-1 in Drosophila:
modifiers of neurodegeneration
24-13
Two genetic screens were performed:
- P-element insertions that disrupt gene function
- EP-element insertions that upregulate expression
The researchers then looked for suppressors or
enhancers of the abnormal eye phenotype
 Hsc70, Hsp70 (disruption makes phenotype worse)
 DnaJ-1 (EP411) - overexpression improves phenotype
 ubiquitin (P1666) and Ub c-terminal hydrolase
(P1779) (disruption makes phenotype worse)
 ub conjugating enzyme (P1303; disruption makes
worse)
 Glutathione-S-Transferase (GST) (2 types)
 involved in detoxification, in particular products
of chemical and oxidative stress
 heat-shock response factor (P292; disruption makes
phenotype worse)
 hsr-omega is a noncoding transcript that is stressinducible and through an unknown mechanism, is
involved in stress adaptation
A convincing association between the
control of protein synthesis and high levels
of heat tolerance in laboratory-selected
lines was first demonstrated in the early
1980s by Alahiotis and Stephanou (1982)
and Stephanou et al. (1983). In these
studies the kinetics of protein synthesis
that was assessed in ovarian tissues
following a heat shock was associated
with changes in the timing and extent of
HSP production, with timing and extent of
housekeeping protein shutdown, and with
heat stress survival differences between
the lines.
Heterogeneous nuclear
ribonucleoproteins
may help explain
general decrease in
protein production
during stress
conditions
Ataxin-1 in Drosophila:
neurodegeneration
24-14
Observation of the first (T1) and second
(T2) thoracic segments of adult Drosophila
interneurons by co-expressing a ventral
nerve cord (VNC) promoter-driven GFP
and control/82Q constructs
 progressive neurodegeneration is
seen in 82Q but not in control
 directly validates the pertinence
of Drosophila model system in
studying human diseases
control
80Q
24-15
Protein degradation diseases:
E3 enzymes implicated
proteasome a target for
several diseases,
including cancer
Process
Substrate (X)
E3
Signal
Transduction
beta-catenin
EGF receptor
SCF
c-Cbl
Transcription
HIF
pVHL
Cell Cycle
Antigen
Processing
p53
cyclins
MHC Class I
antigens
MDM2; E6-AP
SCF; APC
?
?
?
Parkin
Juvenile-onset
familial Parkinson’s
?
?
E6-AP
Angelman’s syndrome
Cancer
EBV
26S
proteasome
CMV
agg’ated
proteins in
general
Alzheimer’s
Adapted from Mayer et al. (2000)
Nature reviews 1, 145-148.
 CANCER
Protein degradation diseases:
examples
24-16
 VHL; most common cause for kidney cancers; component of a a ubiquitin ligase; 100’s of
mutations are known in ~250 amino acid coding region; its biogenesis itself requires CCT
 other
 Angelman’s syndrome
 a mutated E3 enzyme (E6-AP) is associated with this developmental neurological disorder
 VIRAL infections
 in two separate cases, different virus affect the proteolytic degradation machinery (EBV
inhibits the proteasome directly) and antigen processing (CMV)
 Alzheimer’s
 protein aggregates are linked to progressive neurodegeneration; in one case, a frameshift
mutation in a ubiquitin gene appears to cause the disease
 Itch locus
 the Itch gene in mice encodes a novel E3 ligase; disruption of Itch causes a variety of
syndromes that affect the immune system, inflammation of skin gland which result in severe and
constant itching and scarring, etc.
 Liddle syndrome (abnormal kidney function, with excess reabsorption of sodium and loss of potassium from the renal tubule)
 Nedd4 is a ubiquitin protein ligase that binds ENaC subunits (epithelial sodium channel);
mutation in ENaC result in altered homeostasis and hypertension
24-17
ENaC-Nedd4 structure:
clues to Liddle syndrome
 Nedd4 has a HECT ubiquitin ligase domain
 Nedd4 binds ENaC by association of its WW
domain with so-called PY motifs (XPPXY)
 the PY motif(s) is deleted or mutated in
ENaC in Liddle syndrome
 both the tyrosine residue (Y) and the first
proline residue (XP) bind in a groove
solution (NMR) structure
of ENaC peptide TLPIPGTPPPNYDSL
XPPXY
bound to Nedd4
 regulation of the interaction between ENaC
and Nedd4 may affect its turnover (it is shortlived)
 this turnover may be critical to its
function the cell, which is to affect cellular
sodium levels in epithelial cells
Kanelis et al. (2001) Nat. Struct. Biol. 5, 407-412.