A mechanism of lysosomal Calcium entry

溶酶体钙进入机制

• 批准号: 10020204
• 负责人: Yamuna Krishnan
• 金额: $ 22.41万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10020204
• 关键词: ATP phosphohydrolase; Affect; Affinity; Alanine; Amino Acids; Animals; Bioinformatics; Biological; Biological Assay; Brain; Ca(2+)-Transporting ATPase; Calcium; Cell physiology; Cells; Cytosol; DNA; Data; Defect; Disease; Dyes; Endoplasmic Reticulum; Family member; Functional disorder; Homology Modeling; Human; Image; In Situ; Ions; Kinetics; Knowledge; Laboratories; Lead; Location; Lysosomes; Mammalian Cell; Maps; Measures; Mediating; Methods; Modeling; Molecular; Monitor; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Organelles; Parkinson Disease; Parkinsonian Disorders; Pathogenicity; Photobleaching; Positioning Attribute; Protein Import; Reporter; Research; Risk Factors; Spastic Paraplegia; Specificity; Structural Models; Structure; Structure-Activity Relationship; Technology; Therapeutic; Time; Variant; analog; base; cell type; insight; member; mutant; nervous system disorder; novel; predictive modeling; prototype; real-time images; risk variant; small molecule; stem; success; theories; therapeutic target

Lysosomes are highly acidic organelles that integrate important cellular processes in all cell types. There is a preponderance of risk genes for neurological disorders associated with lysosome dysfunction. In the lysosome, lumenal Ca2+ is critical to function, and defects in every known lysosomal Ca2+ release channel leads to a distinct neurological disorder. Yet, dysregulated lysosomal Ca2+ can also arise due to defective import. While much more known of mechanisms that release lysosomal Ca2+, there is a paucity of information on the pathophysiology of Ca2+ import. Notably, the only known lysosomal Ca2+ importer in animals, the P-type ATPase ATP13A2, was recently discovered by my laboratory using newly developed reporter technology for lysosomal Ca2+ imaging. This importer was previously identified as a major risk gene for Parkinson's disease. Thus, a structural level understanding of how by ATP13A2 imports Ca2+ into the lysosome is highly significant. The premise of this proposal is that ATP13A2 function is mechanistically similar to that of SERCA but with lower affinity and/or efficiency of Ca2+ transport. This premise is based on unpublished data from my laboratory using homology modeling, which predicts very high similarity between ATP13A2 and SERCA. SERCA (Sarco/Endoplasmic Reticulum Ca2+ ATPase) is one of the best studied P2-type ATPases. In contrast, ATP13A2 is a P5-type ATPase, an ATPase sub-class yet to be mechanistically characterized. The steps outlined in this proposal will identify and study the molecular mechanism of how ATP13A2 drives lysosomal Ca2+ import by mapping lysosomal Ca2+ dynamics in real-time in live mammalian cells. We plan to create and characterize a photostable lysosomal Ca2+ reporter and develop an assay to map the kinetics of lysosomal Ca2+ import in situ in live cells. Preliminary data shows that we have identified the relevant molecular components to make this photostable organellar Ca2+ reporter. Further, an initial bioinformatics analysis and homology modeling has revealed a remarkable similarly between ATP13A2 and SERCA. This will allow us to pinpoint residues important to Ca2+ import by a P5-type ATPase. By expressing various ATP13A2 mutants and using real-time Ca2+ mapping, we shall be able to identify residues critical to the function of ATP13A2. Successful completion of this research will identify how a major risk gene for Parkinson's disease imports lysosomal Ca2+ and elucidate the first structure-activity relationship in P5-type ATPases. Also, by providing the first practical technology to quantitatively map lysosomal Ca2+ fluxes in live cells (in real-time), we will be in a position to study new lysosomal Ca2+ importers and existing lysosome Ca2+ release channels connected to various neurodegenerative diseases.

溶酶体是高度酸性细胞器，它们整合了所有细胞类型中的重要细胞过程。 与溶酶体功能障碍相关的神经系统疾病有大量的风险基因。 在溶酶体中，Lumenal Ca2+对功能至关重要，并且每个已知的溶酶体Ca2+释放中的缺陷 渠道导致独特的神经系统疾病。但是，由于 导入有缺陷。虽然更了解释放溶酶体Ca2+的机制，但却很少 有关CA2+进口病理生理学的信息。值得注意的是，唯一已知的溶酶体Ca2+进口商 动物是P型ATPase ATP13A2，我的实验室最近使用新开发的动物发现了动物 用于溶酶体CA2+成像的记者技术。此进口商以前被确定为主要风险 帕金森氏病的基因。因此，对ATP13A2如何将Ca2+进口到如何进口到ATP13A2中的结构层面 溶酶体非常重要。 该提案的前提是ATP13A2函数在机械上与Serca相似，但是 Ca2+运输的亲和力较低和/或效率。此前提是基于我的未发表的数据 实验室使用同源性建模，该实验室预测ATP13A2和SERCA之间的相似性非常高。 SERCA（SARCO/内质网Ca2+ ATPase）是研究最佳的P2型ATPase之一。在 对比度，ATP13A2是P5型ATPase，ATPase子类尚未机械表征。 该提案中概述的步骤将识别和研究ATP13A2的分子机制 通过在实时哺乳动物细胞中实时绘制溶酶体Ca2+动力学来驱动溶酶体Ca2+导入。 我们计划创建和表征一个光溶剂体CA2+记者，并开发用于映射的测定法 活细胞中溶酶体Ca2+进口的动力学动力学。初步数据表明，我们已经确定了 相关的分子成分，以使该光稳定器细胞器Ca2+报告基因。此外，最初 生物信息学分析和同源性建模揭示了ATP13A2之间的同样出色 和Serca。这将使我们能够通过P5型ATPase对CA2+导入重要的残留物。经过 表达各种ATP13A2突变体并使用实时CA2+映射，我们将能够识别 残基对ATP13A2的功能至关重要。 成功完成这项研究将确定帕金森氏病的主要风险基因如何 进口溶酶体Ca2+，并阐明了P5型ATPases中的第一个结构活性关系。另外，由 提供第一个实用技术，用于定量绘制活细胞中的溶酶体Ca2+通量（实时）， 我们将可以研究新的溶酶体CA2+进口商和现有的溶酶体CA2+释放通道 连接到各种神经退行性疾病。