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

基本信息

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
项目类别：
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

项目摘要

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 连接酶是一个对蛋白质稳态和信号转导至关重要的蛋白质超家族。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 Parkin 的调节。帕金是控制性破坏的核心通过自噬（线粒体自噬）损伤线粒体。受控线粒体自噬对于心脏尤其重要和神经元健康。 Parkin 突变导致的不受控制的线粒体自噬显然是早发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。这里的中心目标是建立对物理的理解 Parkin 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动，从而导致线粒体自噬和其他生物学缺陷控制不当。尽管人们对帕金功能的生物学和结构基础了解甚多，但仍知之甚少。其调节的物理基础。 Parkin 活性通过其分子内关联而受到抑制泛素样结构域，并通过磷酸化泛素 (pUb) 的结合而变构激活。 Ubl 结构域的磷酸化也会促进激活。这种复杂的监管交叉点只有通过对基础热力学的严格剖析才能理解机制。没有这一知识无法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知识基础其功能可解决人们对 Parkin 变构调节热力学知之甚少的问题。基础帕金的监管控制将被用现代统计热力学描述来描述蛋白质整体。变构调节剂和翻译后修饰的影响将是通过质谱和核磁共振波谱监测的综合氢交换进行检查；先进的核磁共振弛豫技术；单分子荧光；量热法；酶学；和诱变。对 Parkin 监管的更严格和完整的理解将有助于做出强有力的解释的病理突变。并非所有病理突变都可以简单地解释为破坏性突变直接影响催化位点的蛋白质或突变的水平。常见病理例子将检查突变以揭示其对帕金调节保真度影响的基础，并且具有更长的- 确定如何通过小分子干预减轻这种影响的目标。