Chemical Biology of Ubiquitin- and Neddylation-Protein Ligases in Neurodegeneration

神经变性中泛素和 Neddylation 蛋白连接酶的化学生物学

• 批准号: 9808601
• 负责人: Dewey G McCafferty
• 金额: $ 41.44万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808601
• 关键词: Binding; Biochemical; Bioinformatics; Biological Assay; Biology; Biophysics; Biotinylation; Bradykinesia; Brain region; Caenorhabditis elegans; Cell Death; Cell model; Cell physiology; Cells; Characteristics; Chemicals; Complement; Complex; Coupled; Coupling; Data; Deep Brain Stimulation; Defect; Deuterium; Development; Disease; Dopamine; Dyskinetic syndrome; Enzymes; Event; Experimental Models; Exposure to; Generations; Genes; Genetic; Genetic Polymorphism; Goals; Hydrogen; In Situ; Investigation; Kinetics; Lead; Ligands; Ligase; Locomotion; Mass Spectrum Analysis; Methods; Modeling; Molecular; Molecular Abnormality; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotransmitters; Parkinson Disease; Pathology; Peptide Hydrolases; Peroxidases; Phenotype; Point Mutation; Proteins; Proteomics; Regulation; Replacement Therapy; Rest Tremor; Role; SNCA gene; Series; Specificity; Stimulus; Streptavidin; Structure; Substantia nigra structure; Substrate Specificity; Therapeutic; Time; Toxic effect; Ubiquitin; Ubiquitination; Yeasts; alpha synuclein; alpha synuclein gene; ascorbate; base; bioinformatics tool; biophysical properties; dopaminergic neuron; experimental study; genetic risk factor; human pluripotent stem cell; in vitro Assay; insight; knock-down; mutant; neuron loss; neurotoxicity; new therapeutic target; novel; overexpression; parallel computer; protein aggregation; proteostasis; response; screening; small molecule; small molecule inhibitor; tool; ubiquitin ligase; ubiquitin-protein ligase

Parkinson's disease is a neurodegenerative disorder that results from significant and selective loss of neurons in targeted brain regions, specifically dopaminergic neurons within in the brain region Substantia nigra. Neurodegeneration in PD results in locomotion defects such as bradykinesia, dyskinesia, and resting tremors. Although advances in identifying genetic risk factors for the neuronal pathology of the disease are being made, a complete picture of the cellular networks coupling these genetic abnormalities to neurotoxicity and neurodegenerative disease pathology remains a mystery. The long-term goal of this R21 proposal is to interrogate the effects of the small molecule NAB2 on the activity and specificity of the E3 ubiquitin ligase Nedd4 and to illuminate the molecular basis behind NAB2 alleviation of α-synuclein-associated toxicity. In Aim 1 we will examine the ability of the Nedd4 mechanism and kinetics to be altered by chemical modulation will be interrogated through parallel computational, biophysical, and biochemical experiments. The binding mode of NAB2 will be validated using hydrogen-deuterium exchange mass spectrometry, revealing how the ligand engages its target. The biophysical characterization of NAB2 binding will be complimented by a series of mechanistic, in vitro assays which will interrogate Nedd4 function including single- and multi-turnover kinetics, alleviation of autoinhibition, and determination of mechanism (i.e. processive vs. distributive ubiquitylation). The proposed experiments will employ a set of semi-synthetic chemical probes to allow for direct assay of Nedd4 activity. Together, these experiments will determine the mode of action of NAB2 at an enzymatic level and will demonstrate how a small molecule can tune the enzymatic activity of Nedd4, further unraveling the nuances of its mechanism potential for regulation by small molecules. In Aim 2 we will examine the role of Nedd4 in the α- synuclein toxicity network, we will conduct parallel bioinformatic and proximity-driven proteomics analyses to characterize the Nedd4 interactome. Bioinformatic tools will be employed to annotate characteristic structural motifs in the known Nedd4 interactome and α-synuclein toxicity network, determining the predictability of Nedd4 substrates based on structural analyses and identifying putative Nedd4 substrates in the α-synuclein toxicity network. This will be complemented by proximity-driven proteomics using the ascorbate peroxidase (APEX) mass spectrometry platform. In a relevant cellular model, Nedd4 will be expressed as a fusion with APEX, allowing for in situ biotinylation of interacting proteins. Subsequent streptavidin-based purification and protease digest coupled mass spectrometry will allow for characterization of the Nedd4 interactome. This model can be used for analysis +/- NAB2 and +/- toxicity, allowing for differential analysis of the Nedd4 interactome in response to chemical and cellular stimuli. Together, these experiments provide insight into the tunability of Nedd4 as an agent in the rescue of cellular toxicity.

帕金森病是一种神经退行性疾病，是由于神经元显着且选择性丧失所致 目标大脑区域，特别是大脑黑质区域内的多巴胺能神经元。 PD 中的神经变性会导致运动缺陷，例如运动迟缓、运动障碍和静止性震颤。 尽管在识别该疾病的神经元病理学遗传风险因素方面正在取得进展，但 细胞耦合网络的完整图像这些遗传异常对神经毒性和 神经退行性疾病的病理学仍然是一个谜。 R21 提案的长期目标是 探讨小分子 NAB2 对 E3 泛素连接酶 Nedd4 的活性和特异性的影响 并阐明 NAB2 减轻 α-突触核蛋白相关毒性背后的分子基础。 将检查 Nedd4 机制和动力学通过化学调节的能力 通过并行计算、生物物理和生化实验询问结合模式。 NAB2 将使用氢-氘交换质谱法进行验证，揭示配体如何 NAB2 结合的生物物理特征将得到一系列的补充。 机械体外测定将询问 Nedd4 功能，包括单周转动力学和多周转动力学， 缓解自身抑制，并确定机制（即进行性泛素化与分布性泛素化）。 拟议的实验将采用一组半合成化学探针来直接测定 Nedd4 这些实验将共同确定 NAB2 在酶水平上的作用模式，并将 展示小分子如何调节 Nedd4 的酶活性，进一步揭示 在目标 2 中，我们将研究 Nedd4 在 α- 中的作用。 突触核蛋白毒性网络，我们将进行并行生物信息学和邻近驱动的蛋白质组学分析 表征 Nedd4 相互作用组将用于注释特征结构。 已知 Nedd4 相互作用组和 α-突触核蛋白毒性网络中的基序，决定了 Nedd4 的可预测性 基于结构分析的底物并鉴定 α-突触核蛋白毒性中假定的 Nedd4 底物 使用抗坏血酸过氧化物酶（APEX）的邻近驱动蛋白质组学将对此网络进行补充。 在相关的细胞模型中，Nedd4将被表达为与APEX的融合体， 允许随后进行基于链霉亲和素的纯化和蛋白酶的原位生物素化。 消化耦合质谱法将允许表征 Nedd4 相互作用组。 用于分析 +/- NAB2 和 +/- 毒性，允许对 Nedd4 相互作用组进行差异分析 对化学和细胞刺激的反应一起，这些实验提供了对化学和细胞刺激的可调性的深入了解。 Nedd4 作为细胞毒性救援剂。