Defining the mechanistic basis of a prion disaggregase

定义朊病毒解聚酶的机制基础

• 批准号: 8974843
• 负责人: James Shorter
• 金额: $ 29.17万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974843
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Amyloid; Amyloid Proteins; Animals; Architecture; Bacteria; Binding; Biochemical; Biological Assay; Biological Products; Biology; Biophysics; Client; Collaborations; Conflict (Psychology); Cryoelectron Microscopy; Data; Disease; Disease model; Disulfides; Enzymes; Health; Homologous Gene; Human; Individual; Investigation; Link; Methods; Modality; Modeling; Molecular Chaperones; Nature; Nerve Degeneration; Neurodegenerative Disorders; Nucleotides; Parkinson Disease; Patients; Pharmacologic Substance; Plague; Prions; Production; Protein Biochemistry; Protein Footprinting; Proteins; Rattus; Recombinant Proteins; Recombinants; Research; Roentgen Rays; Scanning; Solubility; Stress; Structural Genes; Structure; Substantia nigra structure; System; Therapeutic Agents; Therapeutic Studies; Variant; Yeasts; alpha synuclein; amyloid formation; base; conformer; dimer; in vivo; meetings; monomer; prion-based; protein aggregate; protein aggregation; protein folding; protein misfolding; protein purification; stoichiometry; sup35; therapeutic protein; yeast protein

DESCRIPTION (provided by applicant): The research objective of this proposal is to define the mechanistic basis of Hsp104 a prion disaggregase, which is unknown. Hsp104 is a hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which is the only cellular factor known to rapidly disassemble amyloid or prion fibrils as well as their toxic soluble oligomeric precursors. This catalytic amyloid-disaggregase activity is remarkable because cross-β amyloid is one of the most stable protein-based structures in nature. Moreover, this activity is unique to Hsp104 and is not achieved by any other (AAA+) protein, including ClpX. Even the bacterial Hsp104 homologue, ClpB, which can disaggregate environmentally denatured protein aggregates (like Hsp104), cannot disaggregate amyloids or prions. This Hsp104 activity has enabled yeast to harness multiple prions for beneficial purposes. By contrast, in humans (which curiously lack a direct Hsp104 homologue) prions, amyloids and toxic soluble oligomers cause several fatal neurodegenerative disorders, including Parkinson's disease (PD). Protein aggregation and amyloid formation also plague the purification of recombinant proteins for basic studies and therapeutic purposes. Thus, Hsp104 offers an unparalleled opportunity to: eradicate amyloid (and toxic soluble oligomers)~ understand how amyloid (and toxic soluble oligomers) can be disaggregated~ and understand how AAA+ architecture has been adapted for this modality. Hsp104 could even be specifically enhanced and developed as: (a) an agent to increase protein solubility in diverse expression systems to enable facile purification of recalcitrant proteins for structural and mechanistic studies, and valuable therapeutic proteins for patients~ and (b) a potential therapeutic agent and mechanistic probe for diverse protein-misfolding disorders. Indeed, we have established Hsp104 as the only cellular factor known to dissociate α-synuclein (α-syn) oligomers and amyloids and rescue α-syn-induced neurodegeneration in the substantia nigra of a rat PD model. To develop these potential Hsp104 utilities further, it is critical to understand Hsp104 mechanism, which despite intense investigation remains poorly defined. It is unknown how Hsp104 monomers collaborate within the hexamer to promote prion or protein disaggregation or how Hsp104 engages and eradicates prions. It is also unknown how conformational changes in Hsp104 hexamers facilitate function. We have established key assays and collaborations with leading experts to meet these challenges. Based on our preliminary data, we hypothesize that hexamer plasticity enables Hsp104 to adapt distinct mechanisms to dissolve diverse aggregated structures, including prions. Here, we will combine pure protein biochemistry, biophysics and yeast biology to define the mechanistic basis of Hsp104's amyloid- disaggregase activity via three specific aims: (1) Define how individual subunits of Hsp104 hexamers collaborate to enable protein disaggregation~ (2) Define how Hsp104 engages and deconstructs Sup35 and Ure2 prions~ and (3) Define conformational changes of the Hsp104 hexamer during its ATPase cycle.

描述(申请人提供)：这项建议的研究目标是确定Hsp104是一个未知的Pron解聚酶的机制基础。Hsp104是酵母中的一种六聚体AAA+(与多种活性相关的ATPase)蛋白，是目前已知的唯一能快速分解淀粉样蛋白或蛋白纤维及其有毒的可溶性寡聚体前体的细胞因子。这种催化淀粉样解聚酶的活性是显着的，因为交叉β淀粉样蛋白是自然界中最稳定的蛋白质结构之一。此外，这种活性是Hsp104所独有的，并且不能被任何其他(AAA+)蛋白实现，包括ClpX。即使是细菌Hsp104的同源物ClpB也不能分解淀粉样蛋白或蛋白，它可以分解环境变性的蛋白质聚集体(如Hsp104)。Hsp104的这种活性使酵母能够利用多个Pron用于有益的目的。相比之下，在人类(奇怪的是缺乏Hsp104的直接同源物)中，普鲁恩、淀粉样蛋白和有毒的可溶性寡聚体会导致几种致命的神经退行性疾病，包括帕金森氏病(PD)。蛋白质聚集和淀粉样蛋白的形成也困扰着用于基础研究和治疗目的的重组蛋白质的纯化。因此，HSP104提供了一个无与伦比的机会：根除淀粉样蛋白(和有毒的可溶性寡聚体)~了解淀粉样蛋白(和有毒的可溶性寡聚体)是如何分解的~并了解AAA+架构如何适应这种模式。HSP104甚至可以被特别地增强和开发为：(A)一种能够增加蛋白质在不同表达系统中的溶解度的试剂，从而能够方便地纯化用于结构和机制研究的顽固性蛋白质，以及对患者有价值的治疗性蛋白质；(B)一种潜在的治疗药物和机制探针，用于治疗各种蛋白质错误折叠障碍。事实上，我们已经确定HSP104是唯一已知的能够解离α-SYN寡聚体和淀粉样蛋白并挽救由α-SYN诱导的帕金森病大鼠黑质神经变性的细胞因子。为了进一步开发这些潜在的Hsp104实用工具，了解Hsp104的机制是至关重要的，尽管进行了大量的研究，但对Hsp104的机制仍不清楚。目前尚不清楚Hsp104单体如何在六角体内协作促进普恩或蛋白质解聚，也不清楚HSP104如何与普恩结合并根除普恩。Hsp104六聚体的构象变化如何促进功能也是未知的。我们已经建立了关键的分析方法，并与领先的专家合作，以应对这些挑战。根据我们的初步数据，我们假设六角体的可塑性使Hsp104能够适应不同的机制来溶解不同的聚集结构，包括普恩。在这里，我们将结合纯粹的蛋白质生物化学、生物物理学和酵母生物学，通过三个具体目标来定义Hsp104‘S淀粉样解聚酶活性的机制基础：(1)定义Hsp104六聚体的各个亚基如何协作以实现蛋白质解聚；(2)定义Hsp104如何与Sup35和Ure2 Pron结合和解构；(3)定义Hsp104六聚体在其ATPase周期中的构象变化。