A mechanism of lysosomal Calcium entry

溶酶体钙进入机制

基本信息

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
项目类别：
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

项目摘要

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 This premise is based on unpublished data from my laboratory using homology modeling, which predicts very high similarity between ATP13A2 and SERCA.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.

溶酶体是高度酸性的细胞器，整合所有细胞类型中的重要细胞过程。存在大量与溶酶体功能障碍相关的神经系统疾病的危险基因。在溶酶体中，腔内 Ca2+ 对于功能至关重要，并且每种已知的溶酶体 Ca2+ 释放都存在缺陷通道会导致明显的神经系统疾病。然而，溶酶体 Ca2+ 失调也可能是由于进口有缺陷。虽然释放溶酶体 Ca2+ 的机制更为人所知，但仍缺乏关于 Ca2+ 输入的病理生理学信息。值得注意的是，唯一已知的溶酶体 Ca2+ 输入者动物，P 型 ATP 酶 ATP13A2，是我的实验室最近使用新开发的方法发现的溶酶体 Ca2+ 成像报告技术。该进口商此前被认定为重大风险帕金森病的基因。因此，从结构层面了解 ATP13A2 如何将 Ca2+ 导入溶酶体非常重要。该提议的前提是 ATP13A2 功能在机制上与 SERCA 相似，但 Ca2+ 运输的亲和力和/或效率较低。这个前提是基于我未发表的数据实验室使用同源模型，预测 ATP13A2 和 SERCA 之间具有非常高的相似性。 SERCA（肌肉/内质网 Ca2+ ATP 酶）是研究最充分的 P2 型 ATP 酶之一。在相比之下，ATP13A2 是一种 P5 型 ATP 酶，是一种尚未进行机械表征的 ATP 酶亚类。本提案中概述的步骤将确定和研究 ATP13A2 如何进行的分子机制通过在活哺乳动物细胞中实时绘制溶酶体 Ca2+ 动态来驱动溶酶体 Ca2+ 输入。我们计划创建并表征光稳定的溶酶体 Ca2+ 报告基因，并开发一种分析方法来绘制图谱活细胞中溶酶体 Ca2+ 原位输入的动力学。初步数据显示，我们已经确定制作这种光稳定性细胞器 Ca2+ 报告基因的相关分子成分。此外，初始生物信息学分析和同源建模揭示了 ATP13A2 之间的显着相似性和SERCA。这将使我们能够查明对 P5 型 ATP 酶输入 Ca2+ 重要的残基。经过表达各种 ATP13A2 突变体并使用实时 Ca2+ 绘图，我们将能够识别对 ATP13A2 功能至关重要的残基。这项研究的成功完成将确定帕金森病的主要风险基因如何输入溶酶体 Ca2+ 并阐明 P5 型 ATP 酶中的第一个结构-活性关系。另外，通过提供第一个定量绘制活细胞中溶酶体 Ca2+ 通量的实用技术（实时），我们将能够研究新的溶酶体 Ca2+ 输入器和现有的溶酶体 Ca2+ 释放通道与各种神经退行性疾病有关。