Engineering therapeutic TRIM11 disaggregases for Alzheimer's Disease-Related Dementias (ADRDs)

项目摘要 我们的研究目标是工程改造人类三重基序（TRIM）蛋白TRIM11的变体， 增强的减轻α-突触核蛋白（α-syn）错误折叠和突触核蛋白病的毒性的能力。 突触核蛋白病是一种常见的，使人衰弱的神经退行性疾病，困扰着数百万人 包括帕金森氏病、多系统萎缩和路易体痴呆， 阿尔茨海默病相关痴呆[ADRD]）。在所有的突触核蛋白病中，蛋白质稳态网络不能 对抗小突触前蛋白α-syn的错误折叠。alpha-Syn填充了一系列错误折叠的 从可溶性毒性低聚物到能够引发和 重新传播疾病。alpha-Syn纤维聚集成称为Lewy小体的大细胞质内含物， 突触核蛋白病的病理标志不幸的是，没有有效的治疗方法， 共核蛋白病值得注意的是，我们已经发现，人类TRIM11可以预防和逆转 形成α-syn原纤维并降低其体外和体内毒性。然而，我们怀疑TRIM11 变得不堪重负，无法对抗突触核蛋白病中的α-syn错误折叠。事实上， TRIM11的表达在体内部分减轻了α-syn介导的神经变性，但神经保护作用不明显。 仍然发生不完全和显著的神经变性。因此，增强TRIM 11解聚酶的方法 在突触核蛋白病患者的退化神经元中的活性可以逆转α-syn的有害积累，并为突触核蛋白病提供解决方案。我们假设，工程TRIM11与增强 解聚酶活性将能够更有效地分解α-syn的毒性寡聚体和淀粉样蛋白形式， 其可以赋予突触核蛋白病的治疗益处。因此，我们将设计TRIM11， 对神经毒性α-syn构象异构体的解聚酶活性。然后我们将评估这些增强的能力 TRIM11变体拮抗在哺乳动物中由α-syn寡聚体和原纤维引起的α-syn聚集和毒性 哺乳动物的初级神经元为此，我们将追求三个目标：（1）工程增强TRIM11变体， 减轻酵母中的α-syn聚集和毒性;（2）定义分解α-syn的最佳TRIM11变体 （3）定义增强的TRIM11变体，其减轻α-syn聚集， 对原代神经元的毒性拟议的项目建立了一个管道，首先利用 酵母遗传学从突变体库中精确定位最佳TRIM11解聚酶，并以其 在纯蛋白水平和哺乳动物神经元中进行验证。在这些研究结束时，将有一个明确的 将增强的TRIM11变体移入小鼠突触核蛋白病模型的"去/不去"决定， synucleinopathy患者此外，通过揭示TRIM11解聚酶活性如何通过 我们将开始阐明TRIM11的作用机制。

Project Summary Our research objective is to engineer variants of a human tripartite motif (TRIM) protein, TRIM11, with enhanced ability to mitigate alpha-synuclein (alpha-syn) misfolding and toxicity that underlies synucleinopathies. Synucleinopathies are common, debilitating neurodegenerative disorders that afflict millions of people worldwide and include Parkinson's Disease, Multiple System Atrophy and Lewy Body Dementia, an Alzheimer's Disease-Related Dementia [ADRD]). In all synucleinopathies, the proteostasis network fails to counter the misfolding of the small presynaptic protein alpha-syn. alpha-Syn populates a range of misfolded conformers ranging from soluble toxic oligomers to self-templating amyloid fibrils capable of initiating and propagating disease de novo. alpha-Syn fibrils cluster into large cytoplasmic inclusions termed Lewy Bodies, a pathological hallmark of synucleinopathies. Unfortunately, there are no effective therapeutics for synucleinopathies. Remarkably, we have discovered that human TRIM11 can prevent and reverse the formation of alpha-syn fibrils and reduce their toxicity in vitro and in vivo. However, we suspect that TRIM11 becomes overwhelmed and fails to counter alpha-syn misfolding in synucleinopathies. Indeed, elevating expression of TRIM11 partially mitigates alpha-syn-mediated neurodegeneration in vivo, but neuroprotection is incomplete and significant neurodegeneration still occurs. Thus, methods to enhance TRIM11 disaggregase activity in the degenerating neurons of synucleinopathy patients could reverse deleterious accumulation of alpha-syn and offer a solution for synucleinopathies. We hypothesize that engineering TRIM11 with enhanced disaggregase activity will enable more effective disassembly of toxic oligomeric and amyloid forms of alpha-syn, which could confer therapeutic benefits in synucleinopathies. Thus, we will engineer TRIM11 with enhanced disaggregase activity against neurotoxic alpha-syn conformers. We will then assess the ability of these enhanced TRIM11 variants to antagonize alpha-syn aggregation and toxicity elicited by alpha-syn oligomers and fibrils in mammalian primary neurons. To do so, we will pursue three aims: (1) Engineer enhanced TRIM11 variants to mitigate alpha-syn aggregation and toxicity in yeast; (2) Define optimal TRIM11 variants that disassemble alpha-syn fibrils and oligomers in vitro; and (3) Define enhanced TRIM11 variants that mitigate alpha-syn aggregation and toxicity in primary neurons. The proposed project establishes a pipeline that begins by leveraging the power of yeast genetics to pinpoint optimal TRIM11 disaggregases from mutant libraries and culminates with their validation at the pure protein level and in mammalian neurons. By the end of these studies, there will be a clear “go/no go” decision for moving enhanced TRIM11 variants into mouse synucleinopathy models and ultimately synucleinopathy patients. Moreover, by revealing how TRIM11 disaggregase activity can be enhanced by specific mutations we will begin to clarify the mechanism of TRIM11 action.