Defining mechanisms of AAA+ disaggregases

AAA 解聚的定义机制

基本信息

批准号：
10626853
负责人：
James Shorter
金额：
$ 34.16万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/10626853
关键词：
ATP Hydrolysis ATP phosphohydrolase Address Alzheimer&apos s disease related dementia Amyloid Basic Science Binding Biological Assay Biotechnology Cells Collaborations Couples Cryoelectron Microscopy Curiosities Data Deuterium Development Directed Molecular Evolution Disease Disease model Dissociation Engineering Enzymes Eukaryota Exhibits Goals Homologous Gene Human Hydrogen Industry Link Mass Spectrum Analysis Medicine Mitochondria Mutation Nerve Degeneration Nervous System Neurodegenerative Disorders Nucleotides Orthologous Gene Parkinson Disease Pharmacologic Substance Protein Biochemistry Proteins Rattus Research Resolution Secure Shapes Structure Substantia nigra structure Substrate Specificity Technology Therapeutic Therapeutic Agents Variant alpha synuclein amyloidogenesis combat empowerment frontotemporal lobar dementia amyotrophic lateral sclerosis nanomachine novel novel therapeutics protein TDP-43 protein aggregation protein folding protein misfolding translocase

项目摘要

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。我们的研究目标是确定蛋白质的机制基础。解聚集酶 Hsp104 及其部分人类同源物 Skd3（人类 ClpB），人们对它们知之甚少。在非后生真核生物中，Hsp104 将 ATP 水解与捕获的多种蛋白质的解聚结合起来。无序聚集体、前淀粉样蛋白寡聚体和淀粉样蛋白 Hsp104 是已知解离 α- 的唯一因子。突触核蛋白 (α-syn) 寡聚物和淀粉样蛋白与帕金森病 (PD) 相关并可挽救神经退行性变然而，Hsp104 对 α-syn 的活性有限，并且 Hsp104 浓度较高。因此，我们设计了增强的 Hsp104 变体，可以溶解由形成的原纤维。 ?-syn 以及 TDP-43 和 FUS（与肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆症 (FTD)，一种与阿尔茨海默病相关的痴呆症 (ADRD)，可减轻大脑中的神经退行性变后生动物神经系统比 Hsp104 更有效。 Hsp104 变体缺乏底物特异性，容易产生毒性脱靶效应。为了解决这个问题，我们。设计了新的增强 Hsp104 变体，具有最小的脱靶效应和 α-syn 特异性 Hsp104 这些变异体表现出增强的治疗效用。对抗神经退行性疾病并净化易聚集物质的颠覆性技术奇怪的是，Hsp104 没有精确的后生动物直系同源物。值得注意的是，我们发现人类线粒体中 Hsp104 的部分同源物，一种 AAA+ 蛋白称为 Skd3（人 ClpB），具有与增强的 Hsp104 相当的强大蛋白质解聚酶活性尽管取得了这些重要进展，但我们对 Hsp104 和 Skd3 的机制理解仍受到限制首先，我们不了解 Hsp104 如何选择分解底物。尚未开发出针对 ALS/FTD 的 TDP-43 或 FUS 特定变体其次，我们不知道如何实现。因此，Hsp104 受到核苷酸结合域 2 (NBD2) 特异性突变的调节机制。第三，Skd3 的解聚能力尚不清楚。根据我们的初步数据，我们发现：（1）增强的Hsp104。 (2)具体NBD2 突变通过一种新机制增强 Hsp104；(3) Skd3 是一种强大的人类蛋白质解聚酶与 Hsp104 具有广泛的功能和机制相似性，因此，我们将实现三个目标：（1）定义。具有增强的 TDP-43 和 FUS 选择性的 Hsp104 变体；(2) 定义特定的 NBD2 突变是如何发生的；增强 Hsp104 活性；(3) 定义人类 Skd3 AAA+ 的功能、机制和结构通过这种方式，我们将增强对 Hsp104 和 Skd3 的机制的理解。将促进其在生物技术和医学领域重要应用的发展。