Defining the mechanistic basis of a prion disaggregase

定义朊病毒解聚酶的机制基础

• 批准号: 8438661
• 负责人: James Shorter
• 金额: $ 29.24万
• 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/8438661
• 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; 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; Solutions; Stress; Structural Protein; Structure; Substantia nigra structure; System; Therapeutic Agents; Therapeutic Studies; Variant; Yeasts; amyloid formation; base; conformer; dimer; in vivo; meetings; monomer; prion-based; protein aggregate; protein aggregation; protein folding; protein misfolding; protein purification; public health relevance; stoichiometry; sup35; synuclein; 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-? 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 ?-synuclein (?-syn) oligomers and amyloids and rescue ?-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（由申请人提供）：该提案的研究目标是定义HSP104的机械基础，这是未知的。 HSP104是酵母中的六聚体AAA+（与不同活性相关的ATPases（ATPases），这是唯一已知的细胞因子，唯一可以快速拆卸淀粉样蛋白或prion纤维及其有毒的可溶性寡聚前体。由于交叉？淀粉样蛋白本质上是最稳定的基于蛋白质的结构之一。此外，此活动是HSP104独有的，并且不是其他（AAA+）蛋白（包括CLPX）实现的。即使是细菌HSP104同源物，也可以分解环境变性的蛋白质聚集体（如HSP104），也无法分解淀粉样蛋白或prions。这种HSP104活动使酵母能够利用多个小奖来实现有益的目的。相比之下，在人类（奇怪的是缺乏直接的HSP104同源物）中，淀粉样蛋白和有毒的可溶性低聚物会引起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形成还困扰着重组蛋白的纯化，以进行基础研究和治疗目的。因此，HSP104提供了一个无与伦比的机会：根除淀粉样蛋白（和有毒的可溶性低聚物）〜了解如何分解淀粉样蛋白（和有毒的可溶性低聚物），并了解如何将AAA+结构用于这种模式。 HSP104甚至可以专门增强和开发为：（a）一种增加不同表达系统中蛋白质溶解度的药物，以使顽固性蛋白质可轻松纯化用于结构和机械性研究的顽固性蛋白质，以及对患者的有价值的治疗蛋白〜和（b）潜在的治疗剂和潜在的蛋白质蛋白质蛋白质蛋白质蛋白质的蛋白质。实际上，我们已经将HSP104建立为已知分离的唯一细胞因子？ - synuclein（？ - syn）低聚物和淀粉样蛋白和挽救？ - syn诱导的大鼠PD模型底虫中诱导的神经变性。为了进一步开发这些潜在的HSP104实用程序，了解HSP104机制至关重要，尽管进行了强烈的研究，但这些机制的定义仍然很差。 HSP104单体如何在六聚体中进行合作以促进prion或蛋白质分解，或者HSP104如何参与和消除王室，这是未知的。 HSP104六聚体中构象的变化如何促进功能也未知。我们已经与领先的专家建立了关键的测定法和合作，以应对这些挑战。根据我们的初步数据，我们假设六聚体可塑性使HSP104能够适应不同的机制，以溶解包括prions在内的各种聚合结构。在这里，我们将通过三个特定目的结合纯蛋白质生物化学，生物物理学和酵母生物学，以定义HSP104的淀粉样蛋白 - 淀粉样酶 - 分裂酶活性的机械基础：（1）HSP104六鸟酰基的单个亚基来定义如何合作以启用蛋白质的蛋白质分析〜（2）定义HSP104和decons suppoct和une supons and and and and supons and growsions and growsions（2）定义HSP104六聚体在其ATPase周期中的构象变化。