Quality Control

1. Quality Control 23-1 “QC” - folding or degradation? - Hsp90, CHIP, UFD2

2. Quality control: folding or degradation? non-native protein unfolding refolding refolding degradation Native protein peptides, amino acids Native protein 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 Results show that Hsp90 is implicated in the folding/degradation of luciferase (and other ‘typical’ substrates, e.g. kinases) 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. USA93, 14536-41.

5. CHIP: a novel co-chaperone involved in quality control 23-5 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.

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 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). assists folding assists degradation

8. UFD2:a 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, 635–644. (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 >>> 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

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 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.

14. Cellular indigestion 24-5 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 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 (Parkinson’s), Alzheimer’s disease, huntington’s 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. 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- 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 orexposed to proteasome inhibitor arrestprimarily at the G2/M boundary of the cell cycle. To assess theeffect of protein aggregation on the cell cycle, we transfectedHEK 293 cells with GFP, Q25-GFP, or Q103-GFP and analyzed thecells by flow cytometry for GFP fluorescence and DNA content (Fig. 4B). Cells with the highest level of expression ofQ103-GFP had 4n DNA content, indicating arrest in G2. No suchsubpopulation of cells was observed in cells expressing comparablelevels of Q25-GFP or GFP (Fig. 4B). • result: protein aggregation causes cell-cycle arrest

21. Disease prevention:ataxin-1 as an example 24-11 Ataxin-1Human 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) control 30Q 80Q

23. 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 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 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

26. Protein degradation diseases:E3 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 agg’ated proteins in general Cell Cycle p53 cyclins MDM2; E6-AP SCF; APC Antigen Processing MHC Class I antigens ? CMV Alzheimer’s Juvenile-onset familial Parkinson’s ? ? Parkin ? ? E6-AP Angelman’s syndrome Adapted from Mayer et al. (2000) Nature reviews1, 145-148.

27. Protein degradation diseases:examples 24-16 • CANCER • 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

28. ENaC-Nedd4 structure:clues 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.