Diseases caused by mutations in chaperones - α-crystallin, MKKS/BBS6 chaperonin Neurodegenerative...

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Transcript of Diseases caused by mutations in chaperones - α-crystallin, MKKS/BBS6 chaperonin Neurodegenerative...

  • Slide 1
  • Diseases caused by mutations in chaperones - -crystallin, MKKS/BBS6 chaperonin Neurodegenerative diseases - prions, Huntingtons disease Protein misfolding diseases 12-1
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  • 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 more resistant 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 Neurodegenerative disorders: prions 12-2
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  • 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 contains hamster PrP (lacks mouse PrP) contains hamster PrP (lacks mouse PrP) 12-3 Prion transmission characteristics harbours hamster PrPScharbours murine PrPSc
  • Slide 4
  • Feldman et al. (1999) Formation of the VHL-Elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell 4, 1051-1061. 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. Class Presentations 12-4 Johnston et al. (1998) Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143, 1883-98. 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
  • Slide 5
  • Huntingtons 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 Neurodegenerative disorders: Huntingtons disease 12-5
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  • 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 after 8 hours; aggregation assayed as in (A) time course of aggregation time course of aggregation detected by filter trap assay Huntingtons disease: in vitro model system 12-6
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  • 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 M) was incubated with PreScission protease for 5 h as in previous slide: in the absence (A) or presence (B-F) of chaperones (6 M) (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.) Huntingtons disease: in vitro model system 12-7 control DnaK DnaJ Hdj-1 Hsp70/ATP Hsc70/Hdj-1
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  • 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) Huntingtons disease: in vivo yeast model system 12-8
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  • 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 Esfarjani and Benzer (2000) Science 287, 1837. Huntingtons disease: Drosophila model system 12-9 still see aggregates (as with the in vitro studies)
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  • -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 -crystallin genes A and B cause some major ailments: -crystallin and disease 12-10 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 - and -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 -crystallin occurs) Alexanders disease - the neurodegenerative Alexander's disease is characterized by GFAP co-aggregates with -crystallin; GFAP is closely related to desmin
  • Slide 12
  • R120G -crystallin mutant is found in some patients the chaperone activity of the R120G mutant (located in the highly conserved -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 -crystallin GFAP + R120G -crystallin Association of -crystallin (wild-type and mutant) with GFAP at 37C Perng et al. (1999) J. Biol. Chem. 274, 33235. -crystallin-GFAP experiment 12-11
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  • 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 MKKS/BBS6 mutations cause disease 12-12
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  • 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 PEEAA E, equatorial domain A, apical domain I, intermediate domain P, protrusion II 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. MKKS/BBS6 and other BBS alleles 12-13