Structural studies of PARK14

PARK14的结构研究

• 批准号: 9180460
• 负责人: SERGEY KOROLEV
• 金额: $ 22.73万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180460
• 关键词: Active Sites; Affect; Animal Model; Animals; Ankyrin Repeat; Arachidonic Acids; Binding; Binding Sites; Biochemical; Brain; Calcium; Calmodulin; Cardiovascular Diseases; Catalytic Domain; Cellular Assay; Complex; Crystallization; Data; Development; Diabetes Mellitus; Disease; Epitopes; Event; Foundations; Future; Genes; Goals; Grant; Heart; Heavy Metals; Homologous Gene; In Vitro; Individual; Inherited; Knowledge; Laboratories; Length; Link; Lipids; Lysophospholipids; Malignant Neoplasms; Maps; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Muscular Dystrophies; Mutation; Nerve Degeneration; Organ; PLA2G6 gene; Pancreas; Parkinson Disease; Pathway interactions; Phase; Phospholipase; Physiological; Play; Probability; Property; Proteins; Regulation; Regulatory Element; Resolution; Role; Seitelberger' s Disease; Selenium; Selenomethionine; Signal Pathway; Signal Transduction; Structure; Surface; System; Techniques; Tertiary Protein Structure; Testing; Tissues; base; cell type; cofactor; design; electron density; enzyme activity; enzyme mechanism; enzyme structure; improved; in vivo; insight; mutant; nervous system disorder; novel; novel therapeutic intervention; protein function; protein structure

Mutations in the PARK14 gene are strongly associated with a spectrum of neurological disorders, including Parkinson's disease (PD) and infantile neuroaxonal dystrophy (INAD) through currently unknown mechanisms. The product of the PARK14 gene is an intracellular calcium-independent phospholipase (PLA2G6 or iPLA2β), which has been implicated in numerous cellular pathways. The protein has a unique multi-domain structure and its activity is regulated at several levels. We propose to solve the crystal structure of the protein in order to advance the mechanistic understanding of iPLA2β function in the brain and other organs. An atomic resolution structure of the enzyme is critical for understanding the mechanism of its activity, its regulation and its function in physiological and pathological states. It will provide the foundation for future development of novel therapeutic approaches to treating neurological and cardiovascular diseases as well as diabetes, cancer and muscular dystrophy. iPLA2β modulates membrane properties, produces bioactive lipid messengers such as arachidonic acid and lysophospholipids, and regulates store-operated calcium entry in multiple cell types. Calmodulin (CaM) inhibits iPLA2β enzyme activity in the presence of calcium. Several cofactors reverse the inhibition. Numerous additional regulatory mechanisms have been suggested including oligomerization, ATP binding and interaction with other cofactors and proteins. The large number and variety of signaling pathways affected by iPLA2β and the complexity of its macromolecular interactions complicate defining its function in cellular events and the role it plays in neurological disorders. PARK14 mutations are found in all structural domains of the protein. Studies of these mutations provide a unique opportunity to link regulatory elements and the catalytic activity of iPLA2β to specific signaling mechanisms and functions. Structural information about the conformation of surface epitopes, of the active site and membrane and protein recognition interfaces will be indispensible for these studies. The goal of this proposal is to obtain this structural knowledge to significantly advance the understanding of iPLA2β function. We have crystallized the full-length iPLA2β and now propose to improve the diffraction quality of the crystals and to obtain phasing information to solve the crystal structure of the enzyme. Results from the proposed studies, together with data obtained from biochemical assays, cellular systems and animal models from our group and others will move the entire field forward and will be critical for understanding the functional role of iPLA2β in the brain and other organs including heart and pancreas.

PARK14 基因突变与一系列神经系统疾病密切相关， 包括帕金森病 (PD) 和婴儿神经轴突营养不良 (INAD)，目前未知 PARK14 基因的产物是一种细胞内钙非依赖性磷脂酶。 （PLA2G6 或 iPLA2β），该蛋白质与许多细胞途径有关。 我们建议解决晶体结构的多域结构及其活性。 蛋白质的研究，以促进对 iPLA2β 在大脑和其他器官中功能的机制的理解 酶的原子分辨率结构对于理解其活性机制至关重要， 其在生理和病理状态下的调节及其功能将为未来奠定基础。 开发新的治疗方法来治疗神经系统和心血管疾病以及 糖尿病、癌症和肌肉萎缩症。 iPLA2β 调节膜特性，产生生物活性脂质信使，如花生四烯酸 和溶血磷脂，并调节多种细胞类型中钙库操纵的钙进入。 在存在钙的情况下抑制 iPLA2β 酶活性。 已提出其他调节机制，包括寡聚、ATP 结合和相互作用 与其他辅因子和蛋白质一起，受 iPLA2β 影响的信号通路数量众多且种类繁多。 其大分子相互作用的复杂性使其在细胞事件中的功能和作用的定义变得复杂 研究发现 PARK14 突变存在于该蛋白的所有结构域中。 这些突变提供了连接调控元件和 iPLA2β 催化活性的独特机会 有关表面构象的特定信号机制和功能。 活性位点的表位以及膜和蛋白质识别界面对于这些来说是必不可少的 该提案的目标是获得这种结构知识以显着推进。 了解 iPLA2β 功能。 我们已经结晶了全长 iPLA2β，现在建议提高 iPLA2β 的衍射质量 晶体并获得定相信息以解析酶的晶体结构。 拟议的研究，以及从生化测定、细胞系统和动物模型中获得的数据 我们小组和其他人的成果将推动整个领域向前发展，对于理解功能性至关重要 iPLA2β 在大脑和其他器官（包括心脏和胰腺）中的作用。