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.

项目总结。我们的研究目标是确定这种蛋白质的机制基础