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.

帕金森氏病是一种神经退行性疾病，其由帕金森氏病中神经元的显著和选择性损失引起。 靶向脑区域，特别是脑区域黑质内的多巴胺能神经元。 PD中的神经变性导致运动缺陷，例如运动迟缓、运动障碍和静息震颤。 虽然在确定该疾病的神经病理学的遗传风险因素方面取得了进展， 将这些遗传异常与神经毒性联系起来的细胞网络的全貌， 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NAB 2对E3泛素连接酶Nedd 4的活性和特异性的影响 并阐明NAB 2缓解α-突触核蛋白相关毒性的分子基础。在目标1中， 将检查Nedd 4机制的能力，并通过化学调节改变动力学， 通过并行计算，生物物理和生物化学实验进行询问。的结合模式 NAB 2将使用氢-氘交换质谱法进行验证，揭示配体如何与NAB 2结合。 锁定目标NAB 2结合的生物物理特性将通过一系列的 将询问Nedd 4功能的机制性体外测定，包括单转换和多转换动力学， 自身抑制的缓解和机制的确定（即进行性与分布性泛素化）。的 拟议的实验将采用一组半合成化学探针，以允许直接测定Nedd 4 活动总之，这些实验将确定NAB 2在酶水平上的作用模式，并将 展示了小分子如何调节Nedd 4的酶活性，进一步揭示了Nedd 4的细微差别。 它的机制可能由小分子调节。在目标2中，我们将研究Nedd 4在α- 突触核蛋白毒性网络，我们将进行平行的生物信息学和邻近驱动的蛋白质组学分析， 描述Nedd 4相互作用组的特征。生物信息学工具将用于注释特征结构 已知Nedd 4相互作用组和α-突触核蛋白毒性网络中的基序，确定Nedd 4的可预测性 基于结构分析和鉴定α-突触核蛋白毒性中推定的Nedd 4底物 网络这将通过使用抗坏血酸过氧化物酶（APEX）的邻近驱动蛋白质组学来补充 质谱平台。在相关的细胞模型中，Nedd 4将表达为与APEX的融合物， 允许相互作用蛋白质的原位生物素化。随后的基于链霉亲和素的纯化和蛋白酶 消化偶联质谱法将允许表征Nedd 4相互作用组。该模型可 用于分析+/-NAB 2和+/-毒性，允许在细胞内Nedd 4相互作用组的差异分析。 对化学和细胞刺激的反应。总之，这些实验提供了对 Nedd 4作为拯救细胞毒性的试剂。