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）：本提案的研究目的是确定 Hsp104（一种朊病毒解聚酶）的机制基础，目前尚不清楚。 Hsp104 是一种来自酵母的六聚体 AAA+（与多种活性相关的 ATP 酶）蛋白，它是已知的唯一能够快速分解淀粉样蛋白或朊病毒原纤维及其有毒可溶性寡聚前体的细胞因子。这种催化淀粉样蛋白解聚酶活性非常显着，因为交叉？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，任何其他 (AAA+) 蛋白质（包括 ClpX）都无法实现。即使细菌 Hsp104 同源物 ClpB 可以分解环境变性的蛋白质聚集体（如 Hsp104），也不能分解淀粉样蛋白或朊病毒。 Hsp104 的这种活性使酵母能够利用多种朊病毒达到有益的目的。相比之下，在人类（奇怪的是，人类缺乏直接的 Hsp104 同源物）中，朊病毒、淀粉样蛋白和有毒的可溶性低聚物会导致多种致命的神经退行性疾病，包括帕金森病 (PD)。蛋白质聚集和淀粉样蛋白形成也困扰着用于基础研究和治疗目的的重组蛋白的纯化。因此，Hsp104 提供了无与伦比的机会：根除淀粉样蛋白（和有毒可溶性低聚物）~了解淀粉样蛋白（和有毒可溶性低聚物）如何分解~并了解 AAA+ 结构如何适应这种模式。 Hsp104 甚至可以被专门增强和开发为：(a) 一种在不同表达系统中增加蛋白质溶解度的试剂，以便能够轻松纯化顽固蛋白质以进行结构和机制研究，以及对患者有价值的治疗蛋白质 ~ 和 (b) 一种潜在的治疗方法用于多种蛋白质错误折叠疾病的试剂和机制探针。事实上，我们已经将 Hsp104 确定为唯一已知能够解离 β-突触核蛋白 (β-syn) 寡聚物和淀粉样蛋白并挽救大鼠 PD 模型黑质中 β-syn 诱导的神经变性的细胞因子。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，尽管进行了大量研究，但该机制仍不清楚。目前尚不清楚 Hsp104 单体如何在六聚体内协作以促进朊病毒或蛋白质解聚，也不知道 Hsp104 如何参与和根除朊病毒。 Hsp104 六聚体的构象变化如何促进功能尚不清楚。我们已经与领先专家建立了关键的检测方法和合作来应对这些挑战。根据我们的初步数据，我们假设六聚体可塑性使 Hsp104 能够采用不同的机制来溶解不同的聚集结构，包括朊病毒。在这里，我们将结合纯蛋白质生物化学、生物物理学和酵母生物学，通过三个具体目标来定义 Hsp104 淀粉样蛋白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）定义如何Hsp104 参与并解构 Sup35 和 Ure2 朊病毒~并且 (3) 定义Hsp104 六聚体在其 ATP 酶循环中。