The role of the free energy landscape in Parkin's function and dysfunction in health and disease

自由能景观在健康和疾病中帕金功能和功能障碍中的作用

• 批准号: 10356030
• 负责人: A. JOSHUA WAND
• 金额: $ 34.08万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356030
• 关键词: Address; Allosteric Regulation; Binding; Binding Proteins; Biological; Biology; Calorimetry; Cardiac; Cardiomyopathies; Catalytic Domain; Characteristics; Clinic; Clinical; Competence; Complex; Coupling; Data; Defect; Disease; Dissection; Elements; Entropy; Enzymatic Biochemistry; Enzymes; Eukaryota; Exons; Fluorescence; Foundations; Free Energy; Functional disorder; Goals; Health; Heart Diseases; Hydration status; Hydrogen; Intervention; Knowledge; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methods; Micelles; Mitochondria; Modernization; Molecular Conformation; Monitor; Mutagenesis; Mutation; Myocardium; NMR Spectroscopy; Neurologic; Neurons; PINK1 gene; Parkin; Parkinson Disease; Pathologic; Phosphotransferases; Point Mutation; Post-Translational Protein Processing; Process; Proteins; Proxy; Regulation; Regulatory Element; Relaxation; Relaxation Techniques; Repression; Role; Signal Transduction; Site; Structure; Structure-Activity Relationship; Tertiary Protein Structure; Testing; Thermodynamics; Ubiquitin; Water; base; biophysical techniques; disease-causing mutation; early onset; human disease; insight; member; mitochondrial autophagy; molecular recognition; mutant; nervous system disorder; novel; proteostasis; single molecule; small molecule; ubiquitin ligase; ubiquitin-protein ligase

The RING ubiquitin E3 ligases are a superfamily of proteins critical to protein homeostasis and signaling in eukaryotes. Dysfunctions in E3 ligases are implicated in innumerable human diseases. This proposal focuses on the regulation of the ubiquitin E3 ligase Parkin. Parkin is central to the controlled destruction of damaged mitochondria by autophagy (mitophagy). Controlled mitophagy is particularly essential to cardiac and neuronal health. Uncontrolled mitophagy due to mutations in Parkin is clearly a driver of early onset Parkinson's disease (eoPD). Parkin is now implicated in a number of other neurological diseases, cardiomyopathy and in various cancers. The central goal here is to create an understanding the physical basis for regulation of Parkin and how clinically observed mutations promote unregulated activity leading to inadequately controlled mitophagy and other biological defects. Though much is known about the biology and structural basis of Parkin function, very little is certain about the physical basis for its regulation. Parkin activity is suppressed by its intra-molecular association with a ubiquitin-like domain and is allosterically activated by the binding of phosphorylated ubiquitin (pUb). Phosphorlyation of the Ubl domain also promotes activation. This complicated intersection of regulatory mechanisms can only be understood by the rigorous dissection of the underlying thermodynamics. Without this knowledge one cannot fully interpret the effects of mutations that lead to disease. We shall take advantage of the broad foundation of knowledge of the biology of Parkin and structural basis of its function to address the poorly understood thermodynamics of allosteric regulation of Parkin. The basis for regulatory control of Parkin will be cast in a modern statistical thermodynamics description of the protein ensemble. The influence of allosteric regulators and post-translational modifications will be examined by comprehensive hydrogen exchange monitored by mass spectrometry and NMR spectroscopy; advanced NMR relaxation techniques; single molecule fluorescence; calorimetry; enzymology; and mutagenesis. A more rigorous and complete understanding of the regulation of Parkin will enable a robust interpretation of pathological mutations. Not all pathological mutations can be simply explained as mutations that disrupt the levels of protein or mutations that directly impact the catalytic site. Examples of common pathological mutations will be examined to reveal the basis for their effects on Parkin's regulatory fidelity, with a longer- range goal of determining how this impact might be mitigated by small molecule intervention.

RING泛素E3连接酶是蛋白质稳态和信号传导的关键蛋白质超家族， 真核生物E3连接酶的功能障碍与无数人类疾病有关。该提案重点 泛素E3连接酶帕金的调节。帕金是控制破坏 线粒体自噬（mitophagy）。控制性线粒体自噬对心脏 和神经元健康。由于Parkin突变导致的不受控制的线粒体自噬显然是早发性的驱动因素 帕金森病（eoPD）。帕金现在与许多其他神经系统疾病有关， 心肌病和各种癌症。这里的中心目标是创造一个理解物理 帕金调节的基础以及临床观察到的突变如何促进不受调节的活性， 不适当控制的线粒体自噬和其他生物学缺陷。 尽管人们对帕金功能的生物学和结构基础了解很多，但很少有人能确定 其监管的物理基础。帕金活性通过其与一种蛋白质的分子内缔合而被抑制。 泛蛋白样结构域，并通过磷酸化泛蛋白（pUb）的结合被变构激活。 Ubl结构域的磷酸化也促进活化。这种复杂的监管交叉 只有通过对热力学基础的严格剖析才能理解这些机制。没有 这种知识不能完全解释导致疾病的突变的影响。 我们将利用帕金生物学和结构基础的广泛基础知识 它的功能，以解决鲜为人知的热力学变构调节帕金。基础 帕金的调节控制将被投在现代统计热力学描述的 蛋白质系综变构调节剂和翻译后修饰的影响将是 通过质谱和NMR光谱监测的全面氢交换进行检查; 先进的NMR弛豫技术;单分子荧光;量热法;酶学;和 诱变 对帕金规则的更严格和完整的理解将使我们能够做出有力的解释 病理性突变。并非所有的病理性突变都可以简单地解释为 直接影响催化位点的蛋白质或突变的水平。常见的病理性 突变将被检查，以揭示其对帕金的监管保真度的影响的基础，具有较长的- 确定如何通过小分子干预减轻这种影响的范围目标。