Quality Control

1
Quality Control
23-1
QC - folding or degradation? - Hsp90, CHIP, UFD2
2
Quality control folding or degradation?
23-2
3
QC
23-3

• 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

4
Hsp90 in protein triage
23-4

• 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

yeast cells
HSP90-dependent folding
recovery of activity
- HA
(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
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.
5
CHIP a novel co-chaperone involved in quality
control
23-5
Carboxy terminus of Hsp70-Interacting Protein
CHIP

• 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.
6
Function of CHIP
23-6
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, Hsp40-stimulated 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
carboxy-terminal 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.
7
Function of CHIP
23-7
Re-modelling of chaperoneglucocorticoid 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 chaperoneGR
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).
assists folding
assists degradation
8
UFD2a novel family of ubiquitin ligases
23-8

• 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

9
Discovery of UFD2
23-9
Johnson et al. (1995) A proteolytic pathway that
recognizes ubiquitin as a degradation signal. J.
Biol. Chem. 270, 17442-56. (Varshavsky lab)
Koegl et al. (1999) A novel ubiquitination
factor, E4, is involved in multiubiquitin chain
assembly. Cell 96, 635644. (Jentsch 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
After purification of a protein that interacted
with a ubiquitinated GST-ubiquitin 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 gtgtgt eluted
proteins 2, ubiquitinated Ubi-GST extract gtgtgt
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
10
Protein degradation diseases
24-1
Degradation and disease - aggresomes and russell
bodies cellular indigestion - neurodegeneration
and polyglutamine aggregates - others
11
Quality control in the ER
24-2
membrane-bound chaperone
PDI
newly-imported ER protein is quickly glycosylated
glycotransferase folding sensor
soluble lectin/ chaperone
PDI
protein concentration in ER is extremely high
12
Degradation of abnormal ER proteins
24-3

• 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?

13
Aggresomes and Russell bodies
24-4

• abnormal proteins need to be disposed of, or
  else they end up in inclusions
• ER
• Russell bodies
• aggresomes

e.g. IgG chain

• proteins that are cytosolic can also end up in
  aggresomes

• process of aggresome formation depends on
  microtubules (MTs) and MT-based 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
bodiessubregions 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.
14
Cellular indigestion
24-5
black arrowribosome on RB
whitenormal 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
arise from ER-derived aggregated proteins (e.g.,
mutant Ig chains)
Russell bodies
- 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 are bacterial cytosolic
structures that contain misfolded/aggregated
protein, as well as IbpA and IbpB (small Hsp
molecular chaperones)
Inclusion bodies
15
Proteins that form aggregated cellular inclusions
24-6

• 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
  (Parkinsons), Alzheimers disease, huntingtons
  disease, prion disease, etc.
• Bacterial proteins
• inclusion bodies

process of aggregation is the cause
of cytotoxicity?
aggregates themselves is the cause
of cytotoxicity?
abnormal protein
aggregates
16
objective set up a system where one can
monitor the in vivo level of proteasome activity
in a mammalian model for a misfolding disease
17
Molecular mechanism of Disease
24-7
effect of impairing the proteasome system with a
protein that forms aggresomes
GFP

• GFPu is GFP fused to a short degron, or
  degradation signal at the N-terminus
• cells expressing GFPu were designated GFPu-1
• DMSO is the mock-treated cells (the protesome
  inhibitors are all disolved in DMSO)
• result GFP is a degraded by the
  ubiquitin-proteasome system

GFPu
proteasome inhibitors
protease inhibitors
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 GFPu-1 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.
18
24-8
ALLN
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.
GFPu
GFP
flow cytometry
proteasome inhibitor
GFPu
- inhibitor
inhibition of proteasome act.
biochemical assay of proteasome
(chymotrypsin-like activity)
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

19
24-9
cells expressing Flag-F508 CFTR
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- F508-transfected GFPu-1 cells exhibiting
high (top 3) FLAG- F508 expression compared with
GFPu fluorescence in the subpopulation containing
lower (middle 50) FLAG- F508 expression. (C)
GFPu fluorescence, in FLAG- F508-transfected
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 GFPu-1 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 Q103-MYC-transfected GFPu-1
cells.

• result link between protein aggregation and
  inhibition of UPS

20
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, Q25-GFP, 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 hot-spot 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

21
Disease preventionataxin-1 as an example
24-11
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
22
Ataxin-1 in Drosophila the phenotype
24-12
linked to Spinocerebellar ataxia
using strain harbouring the GMR-GAL4
UAS, Upstream Activating Sequence (for expression
in Drosophila eye)
Polyglutamine (CAG) repeats

• 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
• overexpression of 82Q and 30Q cause similar
  phenotypes in mice cerebellum (neurodegeneration)

30Q
80Q
control
23
Ataxin-1 in Drosophilamodifiers 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
  stress-inducible and through an unknown
  mechanism, is involved in stress adaptation

24
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.
may help explain general decrease in protein
production during stress conditions
Heterogeneous nuclear ribonucleoproteins
25
Ataxin-1 in Drosophilaneurodegeneration
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
26
Protein degradation diseasesE3 enzymes
implicated
24-15
proteasome a target for several diseases,
including cancer
Process
Substrate (X)
E3
Signal Transduction
beta-catenin EGF receptor
SCF c-Cbl
Cancer
EBV
Transcription
HIF
pVHL
26S proteasome
aggated proteins in general
Cell Cycle
p53 cyclins
MDM2 E6-AP SCF APC
Antigen Processing
MHC Class I antigens
?
CMV
Alzheimers
Juvenile-onset familial Parkinsons
?
?
Parkin
?
?
E6-AP
Angelmans syndrome
Adapted from Mayer et al. (2000) Nature reviews
1, 145-148.
27
Protein degradation diseasesexamples
24-16

• CANCER
• VHL most common cause for kidney cancers
  component of a a ubiquitin ligase 100s of
  mutations are known in 250 amino acid coding
  region its biogenesis itself requires CCT
• other
• Angelmans 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)
• Alzheimers
• 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

28
ENaC-Nedd4 structureclues to Liddle syndrome
24-17

• 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
• regulation of the interaction between ENaC and
  Nedd4 may affect its turnover (it is short-lived)
• this turnover may be critical to its function
  the cell, which is to affect cellular sodium
  levels in epithelial cells

solution (NMR) structure of ENaC peptide bound
to Nedd4
TLPIPGTPPPNYDSL
XPPXY
Kanelis et al. (2001) Nat. Struct. Biol. 5,
407-412.