Cytoplasmic Ubiquitin-Specific Protease 19 (USP19) Modulates Aggregation of Polyglutamine-Expanded Ataxin-3 and Huntingtin through the HSP90 Chaperone.
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ABSTRACT:
SUBMITTER: He WT
PROVIDER: S-EPMC4726498 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
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Project description:Expansion of a polymorphic polyglutamine segment is the common denominator of neurodegenerative polyglutamine diseases. The expanded proteins typically accumulate in large intranuclear inclusions and induce neurodegeneration. However, the mechanisms that determine the subcellular site and rate of inclusion formation are largely unknown. We found that the conserved putative nuclear localization sequence Arg-Lys-Arg-Arg, which is retained in a highly aggregation-prone fragment of ataxin-3, did not affect the site and degree of inclusion formation in a cell culture model of spinocerebellar ataxia type 3. Addition of synthetic nuclear export or import signals led to the expected localization of ataxin-3 and determined the subcellular site of aggregate formation. Triggering a cellular stress response by heat shock transcription factor DeltaHSF1 coexpression abrogated aggregation in the cytoplasm but not in the nucleus. These findings indicate that native aggregation-prone fragments derived from expanded ataxin-3 may eventually escape the cytoplasmic quality control, resulting in aggregation in the nuclear compartment.
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Project description:The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.
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Project description:Expansion of polyglutamine (polyQ) tract may cause protein misfolding and aggregation that lead to cytotoxicity and neurodegeneration, but the underlying mechanism remains to be elucidated. We applied ataxin-3 (Atx3), a polyQ tract-containing protein, as a model to study sequestration of normal cellular proteins. We found that the aggregates formed by polyQ-expanded Atx3 sequester its interacting partners, such as P97/VCP and ubiquitin conjugates, into the protein inclusions through specific interactions both in vitro and in cells. Moreover, this specific sequestration impairs the normal cellular function of P97 in down-regulating neddylation. However, expansion of polyQ tract in Atx3 does not alter the conformation of its surrounding regions and the interaction affinities with the interacting partners, although it indeed facilitates misfolding and aggregation of the Atx3 protein. Thus, we propose a loss-of-function pathology for polyQ diseases that sequestration of the cellular essential proteins via specific interactions into inclusions by the polyQ aggregates causes dysfunction of the corresponding proteins, and consequently leads to neurodegeneration.
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Project description:Spinocerebellar ataxia type 1 (SCA1) is one of a group of nine expanded CAG repeat diseases, in which polyglutamine (polyQ) expansion above a threshold is associated with increased disease risk and aggregation. SCA1 is unique in which the polyQ in the disease protein, ataxin1, often contains a few His residues that appear to block toxicity. Here, we ask how His insertions affect aggregation by comparing a Q(30) peptide with and without a centrally inserted His-Gln-His sequence. We found that at pH 7.5-8.5, His interruptions decrease polyQ aggregation rates but do not change the spontaneous growth mechanism: nucleated growth polymerization with a critical nucleus of one without non-fibrillar intermediates. The decreased aggregation rates are because of reductions in nucleation equilibrium constants. At pH 6, however, the His-interrupted peptide aggregates by a different mechanism that involves a low ThT-binding intermediate and produces a polymorphic amyloid product. In aggregates grown at pH 7.5, the His residues are solvent-accessible. Aggregates of His-inserted polyQ are good seeds for Q(30) elongation, suggesting the potential to recruit polyQ proteins in the cell. Our data are therefore most consistent with His insertions blocking toxicity by suppressing rates and/or altering pathways of spontaneous aggregation.
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Project description:Spinocerebellar ataxia type 7 (SCA7) is characterized by cone-rod dystrophy retinal degeneration and is caused by a polyglutamine [poly(Q)] expansion within ataxin-7, a protein of previously unknown function. Here, we report that ataxin-7 is an integral component of the mammalian STAGA (SPT3-TAF9-ADA-GCN5 acetyltransferase) transcription coactivator complex, interacts directly with the GCN5 histone acetyltransferase component of STAGA, and mediates a direct interaction of STAGA with the CRX (cone-rod homeobox) transactivator of photoreceptor genes. Consistent with these results, chromatin immunoprecipitation assays document retinal-specific association of CRX, GCN5, and acetylated histone H3 with CRX target genes. RNA interference studies also implicate ataxin-7 and GCN5 in CRX-dependent gene activation, and histone deacetylase inhibitors restore the compromised expression of a CRX target gene in an ataxin-7-deficient background. Significantly, in relation to SCA7, poly(Q)-expanded ataxin-7 gets incorporated into STAGA and, in a dominant-negative manner, inhibits the nucleosomal histone acetylation function of STAGA GCN5 both in vitro and, based on chromatin immunoprecipitation assays, in SCA7 transgenic mice. These results suggest that the normal function of a poly(Q) disease protein may intersect with its pathogenic mechanism, an observation with significant implications for the molecular basis of all poly(Q) disorders and ultimately for their treatment.
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Project description:Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of <or=1 mM and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ(12)A. 5QMe(2) inhibits not only nucleation of YAQ(12)A but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe(2) acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to beta-sheets. Thus, 5QMe(2) does not disassemble preformed YAQ(12)A; this contrasts with our previously described, backbone N-methylated inhibitors of beta-amyloid aggregation [Gordon, D. J., et al. (2001) Biochemistry 40, 8237-8245; Gordon, D. J., et al. (2002) J. Pept. Res. 60, 37-55]. The mode of action of 5QMe(2) is reminiscent of that of chaperones, because it binds and releases its targets very rapidly and maintains them in a nonaggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by circular dichroism spectroscopy.
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