Defining mechanisms of AAA+ disaggregases

AAA 解聚的定义机制

• 批准号: 10418627
• 负责人: James Shorter
• 金额: $ 34.18万
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10418627
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Alzheimer' s disease related dementia; Amyloid; Basic Science; Binding; Biological Assay; Biotechnology; Cells; Collaborations; Couples; Cryoelectron Microscopy; Data; Deuterium; Development; Directed Molecular Evolution; Disease; Disease model; Engineering; Enzymes; Eukaryota; Exhibits; Goals; Homologous Gene; Human; Hydrogen; Industry; Link; Mass Spectrum Analysis; Medicine; Mitochondria; Mutation; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Nucleotides; Orthologous Gene; Parkinson Disease; Pharmacologic Substance; Protein Biochemistry; Proteins; Rattus; Research; Resolution; Secure; Structure; Substantia nigra structure; Substrate Specificity; Technology; Therapeutic; Therapeutic Agents; Variant; alpha synuclein; amyloidogenesis; base; combat; frontotemporal lobar dementia-amyotrophic lateral sclerosis; nanomachine; novel; novel therapeutics; protein TDP-43; protein aggregation; protein folding; protein misfolding; translocase

Project summary. Our research objective is to define the mechanistic underpinnings of the protein disaggregases, Hsp104, and its partial human homolog, Skd3 (human ClpB), which are poorly understood. In non-metazoan eukaryotes, Hsp104 couples ATP hydrolysis to the disaggregation of diverse proteins trapped in disordered aggregates, preamyloid oligomers, and amyloids. Hsp104 is the only factor known to dissociate α- synuclein (α-syn) oligomers and amyloids linked to Parkinson's Disease (PD) and rescue neurodegeneration in a rat PD model. However, Hsp104 activity is limited against α-syn and high Hsp104 concentrations are required for optimal effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by ?-syn as well as TDP-43 and FUS (which are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), an Alzheimer's Disease-Related Dementia (ADRD), which mitigate neurodegeneration in the metazoan nervous system more effectively than Hsp104. Though potent disaggregases, these potentiated Hsp104 variants lack substrate specificity and are prone to toxic off-target effects. To address this issue, we engineered new potentiated Hsp104 variants with minimal off-target effects and α-syn-specific Hsp104 variants, which exhibited enhanced therapeutic utility. These engineered disaggregases could provide a disruptive technology to combat neurodegenerative disease and enable purification of aggregation-prone proteins for basic or pharmaceutical purposes. Curiously, Hsp104 does not have an exact metazoan ortholog. Remarkably, we have found that a partial homolog of Hsp104 found in human mitochondria, an AAA+ protein called Skd3 (human ClpB), has powerful protein disaggregase activity comparable to potentiated Hsp104 variants. Despite these important advances, our mechanistic understanding of Hsp104 and Skd3 is limited by three critical barriers. First, we do not understand how Hsp104 selects substrates for disaggregation. Thus, we have not yet developed TDP-43- or FUS-specific variants for ALS/FTD. Second, we do not understand how Hsp104 is regulated. Thus, the mechanism by which specific mutations in nucleotide-binding domain 2 (NBD2) potentiate Hsp104 remain unclear. Third, Skd3 is poorly characterized in terms of its disaggregase capabilities, structure, and mechanism. Based on our preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more selective for ALS/FTD-linked TDP-43 and FUS; (2) specific NBD2 mutations potentiate Hsp104 via a novel mechanism; and (3) Skd3 is a powerful human protein disaggregase with broad capabilities and mechanistic similarities to Hsp104. Thus, we will meet three aims: (1) Define Hsp104 variants with enhanced TDP-43 and FUS selectivity; (2) Define how specific NBD2 mutations potentiate Hsp104 activity; (3) Define the capabilities, mechanism, and structure of the human Skd3 AAA+ disaggregase. In this way, we will secure an enhanced mechanistic understanding of Hsp104 and Skd3, which will empower their development for important applications in biotechnology and medicine.

项目摘要。我们的研究目标是定义蛋白质的机械基础 分解HSP104及其部分人同源物SKD3（人类CLPB），它们的理解很少。在 非蛋白酶真核生物，HSP104夫妇ATP水解与被困在 无序的聚集体，前虫类低聚物和淀粉样蛋白。 HSP104是已知分离α-的唯一因素 与帕金森氏病（PD）相关的突触核蛋白（α-Syn）低聚物和淀粉样蛋白 大鼠PD模型。然而，HSP104的活性受到α-Syn的限制，高HSP104浓度为 最佳效果所必需的。那就是我们设计了潜在的HSP104变体，该变体溶解了由 ？-Syn以及TDP-43和FUS（它们与肌萎缩性侧索硬化症（ALS）和额颞 痴呆（FTD），阿尔茨海默氏病与疾病相关的痴呆（ADRD），减轻神经变性 与HSP104相比，后生神经系统更有效。尽管潜在的分类酶，但这些潜在 HSP104变体缺乏底物特异性，并且容易受到有毒的脱靶效应。为了解决这个问题，我们 设计了新的潜在的HSP104变体，具有最小的脱靶效应和α-Syn特异性HSP104 变体，暴露了增强的热实用程序。这些工程化的分类酶可以提供 破坏性的技术来打击神经退行性疾病并实现易于聚集的净化 蛋白质用于基本或药物目的。奇怪的是，HSP104没有确切的后生直系同源物。 值得注意的是，我们发现在人类线粒体（AAA+蛋白）中发现了HSP104的部分同源物 称为SKD3（人CLPB）具有强大的蛋白质分解酶活性，可与电位的HSP104相当 变体。尽管有这些重要的进步，但我们对HSP104和SKD3的机械理解受到限制 三个关键障碍。首先，我们不了解HSP104如何选择分类的基板。那，我们 尚未为ALS/FTD开发TDP-43或FUS特异性变体。第二，我们不明白如何 HSP104受调节。这是核苷酸结合结构域2（NBD2）中特定突变的机制 潜在的HSP104尚不清楚。第三，SKD3的特征在于其分裂酶功能， 结构和机制。根据我们的初步数据，我们假设：（1）潜在的HSP104 可以设计变体对ALS/FTD连接的TDP-43和FUS更具选择性； （2）特定的NBD2 突变潜在的HSP104通过新型机制； （3）SKD3是一种强大的人类蛋白质分类酶 与HSP104具有广泛的功能和机械相似性。那我们将遇到三个目标：（1）定义 具有增强TDP-43和FUS选择性的HSP104变体； （2）定义特定的NBD2突变 潜在的HSP104活性； （3）定义人类SKD3 AAA+的功能，机制和结构 分解酶。这样，我们将获得对HSP104和SKD3的增强机械理解，这 将赋予其在生物技术和医学中重要应用的发展。