Defining mechanisms of AAA+ disaggregases

项目摘要。我们的研究目标是确定蛋白质的机制基础 解聚酶Hsp 104及其部分人类同源物Skd 3（人类ClpB），这些都知之甚少。在 在非后生动物真核生物中，Hsp 104将ATP水解与捕获在 无序聚集体、类淀粉低聚物和类淀粉。Hsp 104是已知的唯一一个解离α- 突触核蛋白（α-syn）寡聚体和淀粉样蛋白与帕金森病（PD）相关，并挽救帕金森病患者的神经变性 大鼠PD模型。然而，Hsp 104对α-syn的活性是有限的，高浓度的Hsp 104对α-syn的活性是有限的。 以达到最佳效果。因此，我们设计了增强的Hsp 104变体，其溶解由 ?- 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），一种阿尔茨海默病相关痴呆症（ADRD），其减轻了大脑中的神经变性。 后生动物神经系统更有效地比Hsp 104。虽然强大的分解，这些增强 Hsp 104变体缺乏底物特异性，并且易于产生毒性脱靶效应。为了解决这个问题，我们 具有最小脱靶效应和α-syn-specific Hsp 104的工程化新增强Hsp 104变体 变体，其表现出增强的治疗效用。这些工程分解气体可以提供一个 颠覆性技术，以打击神经退行性疾病，并使易聚集的纯化 蛋白质用于基本或制药目的。奇怪的是，Hsp 104没有确切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Hsp 104的部分同源物，一种AAA+蛋白， 称为Skd 3（人ClpB），具有与增强的Hsp 104相当的强大的蛋白质解聚酶活性 变体。尽管有这些重要的进展，我们对Hsp 104和Skd 3的机制理解受到限制， 三大关键壁垒首先，我们不了解Hsp 104如何选择底物进行解聚。因此我们 尚未开发出ALS/FTD的TDP-43或FUS特异性变体。第二，我们不明白如何 HSP 104是受调节的。因此，核苷酸结合结构域2（NBD 2）中的特定突变的机制 Hsp 104的作用机制尚不清楚。第三，Skd 3在其解聚能力方面的特征很差， 结构和机制。基于我们的初步数据，我们假设：（1）增强Hsp 104 变体可以被工程化以对ALS/FTD连接的TDP-43和FUS更具选择性;（2）特异性NBD 2 突变通过一种新的机制增强Hsp 104;（3）Skd 3是一种强大的人类蛋白质解聚酶 具有与HSP 104广泛的能力和机制相似性。因此，我们将满足三个目标：（1）定义 具有增强的TDP-43和FUS选择性的Hsp 104变体;（2）定义特异性NBD 2突变 增强Hsp 104活性;（3）明确人Skd 3 AAA+的功能、机制和结构 解聚酶通过这种方式，我们将确保对Hsp 104和Skd 3的机制有更深入的了解， 将使其发展成为生物技术和医学的重要应用。

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.