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

工程治疗 TRIM11 解聚酶治疗阿尔茨海默病相关痴呆症 (ADRD)

• 批准号: 10539674
• 负责人: James Shorter
• 金额: $ 44.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-20 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10539674
• 关键词: Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease related dementia; Amino Acid Motifs; Amyloid; Amyloid Fibrils; Amyotrophic Lateral Sclerosis; Autopsy; Biological Assay; Biotechnology; Cytoplasmic Inclusion; Directed Molecular Evolution; Disease; Engineering; Family; Frontotemporal Dementia; Human; Human Engineering; Huntington Disease; In Vitro; Lewy Bodies; Lewy Body Dementia; Libraries; Mediating; Methods; Modeling; Movement Disorders; Multiple System Atrophy; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathologic; Patients; Persons; Pharmacologic Substance; Proteins; Research; Structure; TRIM Motif; TRIM11 gene; Therapeutic; Therapeutic Agents; Toxic effect; Validation; Variant; Yeasts; alpha synuclein; conformer; effective therapy; in vivo; member; mutant; neuroprotection; neurotoxic; novel; presynaptic; prevent; protein misfolding; proteostasis; synucleinopathy; therapeutically effective; yeast genetics

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.

项目摘要 我们的研究目标是设计一种人类三部分基序(TRIM)蛋白的变体，TRIM11，具有 增强了减轻α-突触核蛋白(α-突触核蛋白)错误折叠和导致突触核病的毒性的能力。 联核病是一种常见的、使人衰弱的神经退行性疾病，困扰着数百万人。 包括帕金森氏症、多系统萎缩和路易体痴呆，以及 阿尔茨海默病相关痴呆[ADRD])。在所有的联核病中，蛋白平衡网络都不能 对抗小突触前蛋白α-syn的错误折叠。Alpha-Syn填充了一系列折叠错误的 从可溶的有毒低聚物到自模板淀粉样纤维的各种构象都能够引发和 新生疾病的传播。Alpha-Syn纤维聚集成大的细胞质内含物，称为路易小体， 是联体核病的病理特征。不幸的是，目前还没有有效的治疗方法 联体核病。值得注意的是，我们发现人类TRIM11可以预防和逆转 在体外和体内形成α-突触蛋白纤维并降低其毒性。然而，我们怀疑TRIM11 变得不堪重负，无法对抗联体核病中的α-SYN错误折叠。的确，提升了 在体内，TRIM11的表达部分减轻了α-突触素介导的神经变性，但神经保护是 不完全的和显著的神经变性仍然会发生。因此，增强TRIM11解聚的方法 突触核病患者变性神经元中的活动可以逆转α-突触蛋白的有害积累，并为突触核病提供解决方案。我们假设工程上的TRIM11具有增强的 解聚酶活性将使有毒的寡聚体和淀粉样α-syn能够更有效地分解， 这可能会在联体核病中带来治疗效果。因此，我们将使用增强版来设计TRIM11 针对神经毒性α-联氨酸异构体的解聚酶活性。然后我们将评估这些增强的能力 TRIM11变异体拮抗α-syn寡聚体和纤维诱导的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)工程师增强的TRIM11变体以 减少酵母中α-SYN的聚集和毒性；(2)确定分解α-SYN的最佳TRIM11变异体 纤维和低聚物在体外；以及(3)定义增强的TRIM11变体，以减轻α-SYN聚集和 对初级神经元的毒性。拟议的项目建立了一条管道，该管道首先利用 酵母遗传学从突变文库中定位最佳的TRIM11解聚酶并最终以其 在纯蛋白质水平和哺乳动物神经元中的验证。到这些研究结束时，将会有一个明确的 将增强的TRIM11变异体转移到小鼠突触核病模型中并最终 联核症患者。此外，通过揭示TRIM11解聚酶活性如何通过 具体的突变我们将开始阐明TRIM11的作用机制。