Defining the mechanistic basis of a prion disaggregase

定义朊病毒解聚酶的机制基础

• 批准号: 9239262
• 负责人: James Shorter
• 金额: $ 33.93万
• 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 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9239262
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Amyloid; Amyotrophic Lateral Sclerosis; Basic Science; Biochemical Genetics; Biological Assay; Biotechnology; Client; Collaborations; Conflict (Psychology); Couples; Cryoelectron Microscopy; Data; Deuterium; Directed Molecular Evolution; Disease; Disease model; Engineering; Enzymes; Frontotemporal Dementia; Genetic study; Goals; HIV; Human; Hydrogen; Industry; Investigation; Link; Mass Spectrum Analysis; Medicine; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Parkinson Disease; Pharmacologic Substance; Prions; Protein Footprinting; Proteins; Rattus; Research; Resolution; Roentgen Rays; Secure; Structure; Substantia nigra structure; Technology; Therapeutic; Therapeutic Agents; Variant; X-Ray Crystallography; Yeasts; alpha synuclein; amyloidogenesis; base; comparative; design; nanomachine; protein TDP-43; protein aggregation; protein folding; protein misfolding; proteostasis; reconstruction; translocase; unfoldase; yeast protein

Project summary: Our research objective is to define the mechanistic basis of Hsp104, a protein disaggregase and hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which remains poorly understood. Hsp104 couples ATP hydrolysis to the dissolution and reactivation of diverse proteins trapped in disordered aggregates, toxic preamyloid oligomers, amyloids, and prions. Hsp104 is the only factor known to dissociate α-synuclein (α-syn) oligomers and amyloids connected with Parkinson's disease (PD) and rescue α-syn-induced neurodegeneration in the substantia nigra of a rat PD model. However, Hsp104 activity is limited against α-syn and very high Hsp104 concentrations are needed for optimal effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by neurodegenerative disease proteins such as TDP-43, FUS, and -syn, and mitigate neurodegeneration in the metazoan nervous system at concentrations where Hsp104 is inactive. Curiously, Hsp104 is absent from metazoa. Thus, Hsp104 and potentiated variants could represent a disruptive technology to enhance proteostasis to counter neurodegenerative disease and enable purification of irksome, aggregation-prone proteins for valuable basic or pharmaceutical purposes. However, these endeavors are frustrated by a limited mechanistic understanding of Hsp104, which despite intense investigation remains stalled at a low level of resolution. Three critical barriers impede our understanding of Hsp104. First, we do not understand how Hsp104 selects clients for disaggregation, which limits our ability to tailor Hsp104 activity for specific substrates. This issue is pernicious because potentiated Hsp104 variants can have damaging, off-target effects due to promiscuous activity, which could restrict therapeutic or biotechnological applications. Second, Hsp104 sequence space remains largely unexplored. It is unclear whether natural Hsp104 orthologues exist with divergent enhanced or selective activity against neurodegenerative disease substrates. Third, there is no atomic structure of the Hsp104 hexamer and conflicting cryo-electron microscopy reconstructions have confused the field. Based on our preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more substrate specific to avoid damaging off-target effects; (2) natural Hsp104 orthologues exist with enhanced activity against neurodegenerative disease substrates and minimal off-target effects; and (3) large structural changes in Hsp104 hexamers upon ATP hydrolysis drive protein disaggregation. Thus, we will meet three aims: (1) Define potentiated Hsp104 variants with enhanced substrate selectivity; (2) Define conserved and divergent activities of natural Hsp104 orthologues; (3) Define high-resolution structural changes in Hsp104 and potentiated variants that drive protein disaggregation. In this way, we will secure a high- resolution mechanistic view of Hsp104, which will empower the engineering of new Hsp104 nanomachines with selective potentiated activity for key applications in biotechnology and medicine.

项目摘要：我们的研究目标是定义蛋白质HSP104的机械基础 酵母中的分类酶和六聚体AAA+（与潜水活动相关的ATPases）蛋白 仍然很了解。 HSP104夫妇ATP水解潜水员的溶解和重新激活 蛋白质被困在无序聚集体，有毒的前序列异构体，淀粉样蛋白和prions中。 HSP104是 仅与帕金森氏症相关的α-突触核蛋白（α-Syn）低聚物和淀粉样蛋白的唯一因素 疾病（PD）和Recueα-Syn诱导的大鼠PD模型的底虫中的神经退行性。 但是，HSP104的活性受到α-Syn的限制，最佳需要很高的Hsp104浓度 效果。这是我们设计了潜在的HSP104变体，该变体溶解了由神经退行性形成的原纤维 疾病蛋白（例如TDP-43，FUS和-syn）以及减轻后生神经系统中的神经变性 HSP104不活跃的浓度下的系统。奇怪的是，HSP104不存在于Metazoa中。那，HSP104 并且潜在的变体可以代表一种破坏性技术，以增强蛋白质的反应 神经退行性疾病并能够纯化有价值的基本或 药物目的。但是，这些努力对有限的机械理解感到沮丧 HSP104（欲望激烈的调查）仍然停滞在低水平的分辨率下。三个关键障碍 阻碍了我们对HSP104的理解。首先，我们不了解HSP104如何为客户选择客户 分解，这限制了我们针对特定底物量身定制HSP104活性的能力。这个问题是有害的 因为潜在的HSP104变体可能会因滥交活动而具有破坏性的，脱离目标的影响，这 可以限制治疗或生物技术应用。其次，HSP104序列空间在很大程度上保持 未探索。目前尚不清楚天然HSP104直系同源物是否存在不同的增强或选择性 针对神经退行性疾病底物的活性。第三，HSP104没有原子结构 六聚体和冲突的冷冻电子显微镜重建使该领域感到困惑。基于我们 初步数据，我们假设：（1）可以将潜在的HSP104变体设计为更多 特定于底物以避免损坏脱靶效应； （2）天然HSP104直系同源物存在 对神经退行性疾病底物的活性增强和最小的脱靶效应； （3） ATP水解后HSP104六聚体的巨大结构变化驱动蛋白质分解。那， 我们将达到三个目标：（1）定义具有增强底物选择性的增强的HSP104变体； （2）定义 天然HSP104直系同源物的保守和不同活动； （3）定义高分辨率结构变化 在HSP104和驱动蛋白质分解的潜在变体中。这样，我们将确保一个高级 HSP104的分辨率机械视图，它将授权新的HSP104纳米机器的工程 具有针对生物技术和医学中关键应用的选择性潜在活动。