Defining mechanisms of AAA+ disaggregases

项目总结。我们的研究目标是确定蛋白质的机械基础 解聚酶Hsp104及其部分人类同源基因Skd3(人类ClpB)，人们对此知之甚少。在……里面 非后生真核生物的Hsp104将ATP的水解与捕获的不同蛋白质的解聚结合在一起 无序的聚集体、前叶低聚物和淀粉样蛋白。HSP104是唯一已知的解离α的因子- 突触核蛋白(α-SYN)寡聚体和淀粉样蛋白与帕金森病(PD)相关并挽救神经变性 建立大鼠帕金森病模型。然而，HSP104的活性对α-SYN是有限的，高浓度的HSP104是 获得最佳效果所必需的。因此，我们设计了增强的Hsp104变体，它可以溶解由 ？-SYN以及TDP-43和FUS(与肌萎缩侧索硬化症(ALS)和额颞部相关) 痴呆症(FTD)，一种阿尔茨海默病相关痴呆症(ADRD)，可缓解 后生动物神经系统比Hsp104更有效。虽然有很强的解聚力，但它们增强了 Hsp104变异体缺乏底物特异性，容易产生有毒的脱靶效应。为了解决这个问题，我们 设计了新的增强型HSP104变种，具有最小的偏离目标效应和α-SYN特异性HSP104 变种，表现出更强的治疗效用。这些经过改造的解聚体可以提供一种 对抗神经退行性疾病并使易于聚集的纯化成为可能的颠覆性技术 用于基础或制药目的的蛋白质。奇怪的是，HSP104并没有精确的后生动物直系同源物。 值得注意的是，我们发现在人类线粒体中发现的HSP104的部分同源物，一种AAA蛋白质 被称为Skd3(人ClpB)，具有与增强的Hsp104相当的强大的蛋白质解聚酶活性 变种。尽管有这些重要的进展，但我们对Hsp104和Skd3的机械性理解受到以下限制 三个关键障碍。首先，我们不了解Hsp104是如何选择底物进行解聚的。因此，我们 还没有开发出针对ALS/FTD的TDP-43或FUS特异性变体。第二，我们不明白如何 HSP104受调控。因此，核苷酸结合域2(Nbd2)的特定突变的机制 增强Hsp104的作用仍不清楚。第三，Skd3在解聚能力方面表现不佳， 结构和机制。根据我们的初步数据，我们假设：(1)增强了Hsp104 变异体可以设计成对ALS/FTD连接的TDP-43和FUS更具选择性；(2)特异性Nbd2 突变通过一种新的机制增强HSP104；以及(3)Skd3是一种强大的人类蛋白解聚酶 具有广泛的功能和与HSP104相似的机械结构。因此，我们将达到三个目标：(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 增强Hsp104活性；(3)确定人Skd3 AAA的能力、机制和结构 解聚。通过这种方式，我们将确保对Hsp104和Skd3有更好的机械性理解，这是 将推动它们在生物技术和医学方面的重要应用的发展。

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.