Defining the mechanistic basis of a prion disaggregase

项目概述：本课题的研究目的是明确Hsp 104蛋白的作用机制 解聚酶和六聚体AAA+（与多种活性相关的ATP酶）蛋白， 仍然知之甚少。Hsp 104将ATP水解与多种细胞的溶解和再活化偶联， 被困在无序聚集体中的蛋白质、有毒的类淀粉低聚物、淀粉样蛋白和朊病毒。HSP 104是 已知唯一能解离α-突触核蛋白（α-syn）寡聚体和与帕金森氏症相关的淀粉样蛋白的因子 疾病（PD）和救援α-syn诱导的神经变性在大鼠PD模型的黑质。 然而，Hsp 104对α-syn的活性是有限的，并且需要非常高的Hsp 104浓度以获得最佳的活性。 方面的影响.因此，我们设计了增强的Hsp 104变体，它溶解了神经退行性疾病形成的纤维， 疾病蛋白，如TDP-43，FUS和β-syn，并减轻后生动物神经变性 在Hsp 104无活性的浓度下的系统。奇怪的是，Hsp 104在后生动物中并不存在。因此，HSP 104 增强的变体可能代表一种破坏性的技术，以增强蛋白质稳态， 神经退行性疾病，并能够纯化讨厌的、易于聚集的蛋白质，用于有价值的碱性或碱性磷酸酶。 制药目的。然而，这些努力由于对环境的有限的机械理解而受挫。 Hsp 104，尽管进行了深入的研究，但仍然停留在低水平的分辨率上。三大关键障碍 阻碍了我们对HSP 104的理解。首先，我们不明白Hsp 104如何选择客户端， 解聚，这限制了我们为特定底物定制Hsp 104活性的能力。这个问题是有害的 因为增强的Hsp 104变体可能由于混杂活性而具有破坏性的脱靶效应， 可能会限制治疗或生物技术应用。第二，Hsp 104序列空间大部分保留 未开发的目前还不清楚是否存在天然的Hsp 104直向同源物， 抗神经退行性疾病底物的活性。第三，没有HSP 104的原子结构 六聚体和相互冲突的冷冻电子显微镜重建混淆了该领域。基于我们 根据初步数据，我们假设：（1）增强的Hsp 104变体可以被工程化为更多 底物特异性，以避免破坏性脱靶效应;（2）天然Hsp 104直向同源物存在， 增强的抗神经退行性疾病底物的活性和最小的脱靶效应;和（3） Hsp 104六聚体在ATP水解后的大的结构变化驱动蛋白质解聚。因此，在本发明中， 我们将满足三个目标：（1）定义具有增强的底物选择性的增强的Hsp 104变体;（2）定义 天然Hsp 104同源物的保守和差异活性;（3）定义高分辨率结构变化 在热休克蛋白104和增强的变体，驱动蛋白质解聚。这样，我们就能获得一个高- Hsp 104的解析机制视图，这将为新的Hsp 104纳米机器的工程设计提供支持 具有选择性增强的活性，用于生物技术和医学的关键应用。

Project summary: Our research objective is to define the mechanistic basis of Hsp104, a protein disaggregase and hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which remains poorly understood. Hsp104 couples ATP hydrolysis to the dissolution and reactivation of diverse proteins trapped in disordered aggregates, toxic preamyloid oligomers, amyloids, and prions. Hsp104 is the only factor known to dissociate α-synuclein (α-syn) oligomers and amyloids connected with Parkinson's disease (PD) and rescue α-syn-induced neurodegeneration in the substantia nigra of a rat PD model. However, Hsp104 activity is limited against α-syn and very high Hsp104 concentrations are needed for optimal effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by neurodegenerative disease proteins such as TDP-43, FUS, and -syn, and mitigate neurodegeneration in the metazoan nervous system at concentrations where Hsp104 is inactive. Curiously, Hsp104 is absent from metazoa. Thus, Hsp104 and potentiated variants could represent a disruptive technology to enhance proteostasis to counter neurodegenerative disease and enable purification of irksome, aggregation-prone proteins for valuable basic or pharmaceutical purposes. However, these endeavors are frustrated by a limited mechanistic understanding of Hsp104, which despite intense investigation remains stalled at a low level of resolution. Three critical barriers impede our understanding of Hsp104. First, we do not understand how Hsp104 selects clients for disaggregation, which limits our ability to tailor Hsp104 activity for specific substrates. This issue is pernicious because potentiated Hsp104 variants can have damaging, off-target effects due to promiscuous activity, which could restrict therapeutic or biotechnological applications. Second, Hsp104 sequence space remains largely unexplored. It is unclear whether natural Hsp104 orthologues exist with divergent enhanced or selective activity against neurodegenerative disease substrates. Third, there is no atomic structure of the Hsp104 hexamer and conflicting cryo-electron microscopy reconstructions have confused the field. Based on our preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more substrate specific to avoid damaging off-target effects; (2) natural Hsp104 orthologues exist with enhanced activity against neurodegenerative disease substrates and minimal off-target effects; and (3) large structural changes in Hsp104 hexamers upon ATP hydrolysis drive protein disaggregation. Thus, we will meet three aims: (1) Define potentiated Hsp104 variants with enhanced substrate selectivity; (2) Define conserved and divergent activities of natural Hsp104 orthologues; (3) Define high-resolution structural changes in Hsp104 and potentiated variants that drive protein disaggregation. In this way, we will secure a high- resolution mechanistic view of Hsp104, which will empower the engineering of new Hsp104 nanomachines with selective potentiated activity for key applications in biotechnology and medicine.