Protein misfolding diseases

1. Protein misfolding diseases 12-1 • Diseases caused by mutations in chaperones • - α-crystallin, MKKS/BBS6 chaperonin • Neurodegenerative diseases • prions, Huntington’s disease

2. Neurodegenerative disorders: prions 12-2 • pathogenesis of many neurodegenerative disorders is due to abnormal protein conformation • common theme in diseases is conversion of normal cellular and/or circulating protein into an insoluble, aggregated, beta-sheet rich form which is deposited in the brain as an amyloid • deposits are toxic and produce neuronal dysfunction and death • prion-related diseases occur when conversion of a normal prion protein, PrP, into an infectious and pathogenic form, PrPSc (Prion Protein Scrapie). Prion diseases: • Creutzfeld Jacob disease, Kuru, Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia, Scrapie (sheep), Bovine spongiform encephalopathy (BSE or ‘mad cow’), chronic wasting disease (mule deer, elk), feline spongiform encephalopathy • the conversion of PrP into PrPSc is a conformational one; the PrPSc form is moreresistant to proteases and is detergent-insoluble • PrPSc forms amyloid fibrils in the brain; injection of this material into the brains of normal mice leads to disease • the normal function of PrP is unknown; transgenic mice lacking this protein grow normally • Other proteins unrelated in sequence to PrP have similar properties: • e.g., yeast Sup35, Ure2p

3. Prion transmission characteristics 12-3 harbours hamster PrPSc harbours murine PrPSc contains hamster PrP (lacks mouse PrP) contains hamster PrP (lacks mouse PrP) Note: - testing for infectivity with PrPSc is done by injecting brain material from an infected animal into the brain of another animal - transgenic mice devoid of mouse PrP cannot be infected by mouse PrPSc

4. Class Presentations • TRiC stands for TCP-1 Ring Complex and is the same eukaryotic cytosolic chaperonin as CCT (Chaperonin containing TCP-1). • CCT was first thought to assist the folding of only actins and tubulins, but recently, it has been found to bind ~10% of all cell proteins and is known to assist the folding of numerous other proteins, including a viral capsid protein, myosin, luciferase and VHL. Feldman et al. (1999) Formation of the VHL-Elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell4, 1051-1061. • there exists a cellular mechanism by which misfolded/aggregated proteins are sequestered in the cell • such sequestration occurs near the centrosome in an organelle-like structure commonly termed ‘aggresome’ Johnston et al. (1998) Aggresomes: a cellular response to misfolded proteins. J. Cell Biol.143, 1883-98. 12-4

5. Neurodegenerative disorders: Huntington’s disease 12-5 • Huntington’s disease (HD) is a very common syndrome that affects numerous people • It is caused by the expansion of CAG trinucleotide repeats (encoding polyglutamine) within a large protein (350 kDa) termed huntingtin • the function of huntingtin is unclear; evidence points to trafficking (vesicular) • normal and disease forms • unaffected individuals carry between 6 and 39 repeats in exon 1 of huntingtin • HD patients typically have between 36-180 repeats in exon 1 of huntingtin • mutant forms of huntingtin with expanded repeats form nuclear and cytoplasmic aggregates in human brain tissue

6. Huntington’s disease: in vitro model system 12-6 • can express protein fusion with different numbers of CAG repeats and study • Muchowski et al. (2000) PNAS 97, 7841. produced GST-HD proteins (HD20Q and HD53Q) then cleaved off HD from tag using protease that cleaves between GST and HD; aggregation was then followed in the presence or absence of chaperones • found that combination of Hsp40 and DnaK were most effective at preventing aggregation time course of aggregation detected by ‘filter trap’ assay after 8 hours; aggregation assayed as in (A) time course of aggregation

7. Huntington’s disease: in vitro model system 12-7 control Hdj-1 • GST-HD proteins were induced to aggregate by cleavage (as before) in the presence or absence of chaperones • fibrils/aggregate formation was observed by electron microscopy Suppression of HD exon 1 fibril formation by Hsp40 and Hsp70 in vitro. GST-HD fusion protein (3 µM) was incubated with PreScission protease for 5 h as in previous slide: in the absence (A) or presence (B-F) of chaperones (6 µM) (B) DnaK (C) DnaJ (D) Hdj-1 (E) Hsc70/ATP (F) Hsc70/Hdj-1/ATP (Hsc70/Hdj-1 = 2:1). Samples then were analyzed by EM. (Bar = 100 nm.) Hsp70/ATP DnaK DnaJ Hsc70/Hdj-1

8. Huntington’s disease: in vivo yeastmodel system 12-8 • Huntingtin constructs with Exon 1 and containing 20, 39 or 53 CAG repeats as well as a c-myc tag (which is recognized by antibody and can be immunoprecipitated) were expressed in S. cerevisiae • (A) *=SDS-insoluble aggregates that do not penetrate the gel • (A) **=degradation product of full-length protein • (B) filter-trap assay; T=total, S=soluble, P=pellet after centrifugation • (D) immunoprecipitation of different proteins with anti Ssa (cytosolic) and Ssb (ribosome-bound) Hsp70 protein homologues from yeast, as well as anti-Ydj1 (Hsp40 homologue) • High-level expression of Hsp70/40 in yeast with HD53Q made the aggregates SDS-soluble! (not shown)

9. Huntington’s disease: Drosophila model system 12-9 • expressed HA-tagged 127 CAG repeat-protein (127Q) in the eye, causing abnormalities/polyQ deposits • GMR has 5 tandem copies of a response element derived from the rhodopsin 1 gene promoter) • GAL4 is a transcription factor • UAS, ‘Upstream Activating Sequence’ required for GAL4-dependent gene expression • flies carrying GMR-GAL + UAS127Q were crossed with EP-element insertion strains (7000) • screened for suppression or enhancement of toxicity • found: dhdJ1, an Hsp40 homologue; dtpr2 is a TPR-containing protein with J domain • still see aggregates (as with the in vitro studies) Esfarjani and Benzer (2000) Science287, 1837.

10. TRANSGENIC STRAIN CARRYING GMR PROMOTER-GAL4 CONSTRUCT (HIGH-LEVEL EXPRESSION IN EYE) GMR GMR GMR GMR GMR GAL4 CONTRUCT CROSSED INTO THE ABOVE STRAIN (UAS ACTIVATED BY GAL4 TO INDUCE HIGH-LEVEL EXPRESSION OF 127Q) UAS 127Q

11. α-crystallin and disease 12-10 • α-crystallin belongs to the class of molecular chaperones collectively termed small heat-shock proteins • functions include (but is not limited to) maintaining microfilament stability (e.g., intermediate filaments and perhaps actin and tubulin) • present in all tissue types and ubiquitous in the three domains mutations in α-crystallin genes A and B cause some major ailments: • cataracts - function is as a structural protein as well as a molecular chaperone; it makes up nearly 1/3 of the eye lens protein, while β- and γ-crystallins make up close to the other 2/3 • desmin-related myopathy - desmin is an intermediate filament; mutation in the chaperone result in the accumulation of intracellular aggregates of desmin (co-aggregation with α-crystallin occurs) • Alexander’s disease - the neurodegenerative Alexander's disease is characterized by GFAP co-aggregateswith α-crystallin; GFAP is closely related to desmin

12. α-crystallin-GFAP experiment 12-11 • R120G α-crystallin mutant is found in some patients • the chaperone activity of the R120G mutant (located in the highly conserved α-crystallin domain) is not completely lost compared to the wild-type chaperone intact (as judged by prevention-of-aggregation experiments) • reason why the mutant chaperone associates more strongly with GFAP (and desmin) is unclear • specificity of binding causing the problem? GFAP + wt α -crystallin GFAP + R120G α -crystallin Association of α-crystallin (wild-type and mutant) with GFAP at 37ºC Perng et al. (1999) J. Biol. Chem.274, 33235.

13. MKKS/BBS6 mutations cause disease 12-12 • MKKS/BBS6 is one of 12 genes that cause Bardet-Biedl Syndrome (i.e., a polygenic disorder) • mapping of BBS genes relied on screening inbred populations (e.g., Bedoin arabs, Old Order Amish, Newfoundland) • BBS phenotypes: obesity, kidney and liver problems, retinal degeneration, cardiomyopathy, diabetes, mental retardation, anosmia, hearing impairment, polydactyly, etc. • MKKS/BBS6 is related to the chaperonin CCT • two other chaperonin-like genes were found: BBS10, BBS12

14. MKKS/BBS6 and other BBS alleles 12-13 • the chaperonin is related to the eukaryotic cytosolic chaperonin CCT • it is found only in vertebrates and ‘more evolved’ organisms • it is highly divergent although it is clearly a Group II chaperonin E, equatorial domain A, apical domain I, intermediate domain P, protrusion E I A P A I E • BBS4 is involved in microtubule anchoring; it contains multiple TPR motifs: 34 amino acid repeats of helix-loop-helix - TPRs are protein-protein interaction domains • BBS proteins are required for proper cilia function; BBS is therefore a ciliopathy • improper function of ciliary genes results in numerous ailments, including retinal degeneration, polycystic kidneys, skeletal anomalies, etc.