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

帕金森病是一种神经退行性疾病，其原因是脑内神经元的显著和选择性丢失 靶向大脑区域，特别是黑质脑区内的多巴胺能神经元。 帕金森病患者的神经变性会导致运动障碍，如运动迟缓、运动障碍和静止性震颤。 尽管在识别这种疾病的神经元病理的遗传风险因素方面取得了进展，但 将这些遗传异常与神经毒性相结合的细胞网络的完整图像 神经退行性疾病的病理仍是一个谜。这项R21提案的长期目标是 小分子NAB2对E3泛素连接酶Nedd4活性和特异性的影响 阐明NAB_2减轻α-突触核蛋白相关毒性的分子基础。在目标1中，我们 将考察Nedd4的机制和动力学被化学调制改变的能力 通过并行的计算、生物物理和生化实验进行讯问。的绑定模式 NAB2将使用氢-重离子交换质谱学进行验证，揭示配体如何 与目标交火。NAB2结合的生物物理特征将由一系列 将询问Ned4功能的机械性、体外分析，包括单周转和多周转动力学， 减轻自身抑制，并确定机制(即进行性泛素化与分布性泛素化)。这个 拟议的实验将使用一套半合成的化学探针，以允许直接分析Nedd4 活动。总之，这些实验将在酶水平上确定NAB2的作用模式，并将 演示小分子如何调节Nedd4的酶活性，进一步揭开 它的机制有可能被小分子调节。在目标2中，我们将研究Ned4在α中的作用- ，我们将进行平行生物信息学和邻近驱动的蛋白质组学分析，以 描述Nedd4相互作用组。将使用生物信息学工具来注释特征结构 已知的Nedd4相互作用体和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和鉴定α-突触核蛋白毒性中可能的NED4底物 网络。这将得到使用抗坏血酸过氧化物酶(APEX)的邻近驱动蛋白质组学的补充 质谱学平台。在相关的细胞模型中，Nedd4将被表达为与APEX的融合， 允许相互作用蛋白的原位生物素化。基于链霉亲和素的后续纯化和蛋白酶 消化耦合质谱仪将对Nedd4相互作用体进行表征。这个模型可以是 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。综上所述，这些实验提供了对 Nedd4作为一种解救细胞毒性的试剂。

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.