Evidence for a recruitment and sequestration mechanism in Huntington's disease.
ABSTRACT: Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.
Project description:The effect of expressing human huntingtin fragments containing polyglutamine (polyQ) tracts of varying lengths was assessed in Caenorhabditis elegans ASH sensory neurons in young and old animals. Expression of a huntingtin fragment containing a polyQ tract of 150 residues (Htn-Q150) led to progressive ASH neurodegeneration but did not cause cell death. Progressive cell death and enhanced neurodegeneration were observed in ASH neurons that coexpressed Htn-Q150 and a subthreshold dose of a toxic OSM-10::green fluorescent protein (OSM-10::GFP) fusion protein. Htn-Q150 huntingtin protein fragments formed protein aggregates in ASH neurons, and the number of ASH neurons containing aggregates increased as animals aged. ASH neuronal cell death required ced-3 caspase function, indicating that the observed cell death is apoptotic. Of interest, ced-3 played a critical role in Htn-Q150-mediated neurodegeneration but not in OSM10::GFP-mediated ASH neurodegeneration. ced-3 function was important but not essential for the formation of protein aggregates. Finally, behavioral assays indicated that ASH neurons, coexpressing Htn-Q150 and OSM10::GFP, were functionally impaired at 3 days before the detection of neurodegeneration, cell death, and protein aggregates.
Project description:The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
Project description:The presence of expanded poly-glutamine (polyQ) repeats in proteins is directly linked to the pathogenesis of several neurodegenerative diseases, including Huntington's disease. However, the molecular and structural basis underlying the increased toxicity of aggregates formed by proteins containing expanded polyQ repeats remain poorly understood, in part due to the size and morphological heterogeneity of the aggregates they form in vitro. To address this knowledge gap and technical limitations, we investigated the structural, mechanical and morphological properties of fibrillar aggregates at the single molecule and nanometer scale using the first exon of the Huntingtin protein as a model system (Exon1). Our findings demonstrate a direct correlation of the morphological and mechanical properties of Exon1 aggregates with their structural organization at the single aggregate and nanometric scale and provide novel insights into the molecular and structural basis of Huntingtin Exon1 aggregation and toxicity.
Project description:In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.
Project description:Huntington's disease and several other neurological diseases are caused by expanded polyglutamine [poly(Gln)] tracts in different proteins. Mechanisms for expanded (>36 Gln residues) poly(Gln) toxicity include the formation of aggregates that recruit and sequester essential cellular proteins [Preisinger, E., Jordan, B. M., Kazantsev, A. & Housman, D. (1999) Phil. Trans. R. Soc. London B 354, 1029-1034; Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001) J. Mol. Biol. 311, 173-182] and functional alterations, such as improper interactions with other proteins [Cummings, C. J. & Zoghbi, H. Y. (2000) Hum. Mol. Genet. 9, 909-916]. Expansion above the "pathologic threshold" ( approximately 36 Gln) has been proposed to induce a conformational transition in poly(Gln) tracts, which has been suggested as a target for therapeutic intervention. Here we show that structural analyses of soluble huntingtin exon 1 fusion proteins with 16 to 46 glutamine residues reveal extended structures with random coil characteristics and no evidence for a global conformational change above 36 glutamines. An antibody (MW1) Fab fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs containing normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a "linear lattice" model for poly(Gln), in which expanded poly(Gln) tracts have an increased number of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity of expanded poly(Gln) tracts and a structural framework for drug design.
Project description:This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.
Project description:Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (? 100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.
Project description:Aggregation of proteins containing polyglutamine (polyQ) expansions characterizes many neurodegenerative disorders, including Huntington's disease. Molecular chaperones modulate the aggregation and toxicity of the huntingtin (Htt) protein by an ill-defined mechanism. Here we determine how the chaperonin TRiC suppresses Htt aggregation. Unexpectedly, TRiC does not physically block the polyQ tract itself, but rather sequesters a short Htt sequence element, N-terminal to the polyQ tract, that promotes the amyloidogenic conformation. The residues of this element essential for rapid Htt aggregation are directly bound by TRiC. Our findings illustrate how molecular chaperones, which recognize hydrophobic determinants, can prevent aggregation of polar polyQ tracts associated with neurodegenerative diseases. The observation that short endogenous sequence elements can accelerate the switch of polyQ tracts to an amyloidogenic conformation provides a novel target for therapeutic strategies.
Project description:Expansion of a poly-glutamine (polyQ) repeat in a group of functionally unrelated proteins is the cause of several inherited neurodegenerative disorders, including Huntington's disease. The polyQ length-dependent aggregation and toxicity of these disease proteins can be reproduced in Saccharomyces cerevisiae. This system allowed us to screen for genes that when overexpressed reduce the toxic effects of an N-terminal fragment of mutant huntingtin with 103 Q. Surprisingly, among the identified suppressors were three proteins with Q-rich, prion-like domains (PrDs): glycine threonine serine repeat protein (Gts1p), nuclear polyadenylated RNA-binding protein 3, and minichromosome maintenance protein 1. Overexpression of the PrD of Gts1p, containing an imperfect 28 residue glutamine-alanine repeat, was sufficient for suppression of toxicity. Association with this discontinuous polyQ domain did not prevent 103Q aggregation, but altered the physical properties of the aggregates, most likely early in the assembly pathway, as reflected in their increased SDS solubility. Molecular simulations suggested that Gts1p arrests the aggregation of polyQ molecules at the level of nonfibrillar species, acting as a cap that destabilizes intermediates on path to form large fibrils. Quantitative proteomic analysis of polyQ interactors showed that expression of Gts1p reduced the interaction between polyQ and other prion-like proteins, and enhanced the association of molecular chaperones with the aggregates. These findings demonstrate that short, Q-rich peptides are able to shield the interactive surfaces of toxic forms of polyQ proteins and direct them into nontoxic aggregates.
Project description:Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.