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Calcium homeostasis in organelles

细胞器中的钙稳态

基本信息

批准号：
10202773
负责人：
Yamuna Krishnan
金额：
$ 36.04万
依托单位：
UNIVERSITY OF CHICAGO
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：
2020-07-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10202773
关键词：
Acidity Affect Alanine Animals Binding Binding Sites Biological Assay Caenorhabditis elegans Calcium Catalogs Data Defect Diagnosis Disease Dyes Endoplasmic Reticulum Engineering Genes Genetic Genetic Models Goals Growth Heavy Metals Hepatocyte Homeostasis Homologous Gene Homology Modeling Human Image Impairment Ions Knowledge Laboratories Lead Lysosomes Maps Measures Mediating Metal Ion Binding Metals Methodology Molecular Mutation Analysis Nematoda Neurodegenerative Disorders Normalcy Organelles Organism PARK9 gene Parkinson Disease Pathway interactions Phenotype Proteins Reporter Reporting Saccharomyces cerevisiae Structure System Testing Variant Whole Organism base brain cell experimental study innovation insight molecular modeling mutant nervous system disorder novel overexpression quantitative imaging risk variant screening success theories uptake vacuolar H+-ATPase

项目摘要

Project Summary Defects in many known regulators of lumenal Ca2+ in lysosomes lead to distinct neurological disorders by disrupting lysosomal Ca2+ levels. Lysosomal Ca2+ can be dysregulated by defects in either Ca2+ release channels or transporters. While there are several examples of lysosomal Ca2+ release channels, there is only one example of a protein that facilitates lysosomal Ca2+ import. This solitary example was identified by my lab to be catp-6 in C. elegans. Its human homolog, ATP13A2, is one of the major risk genes for Parkinson's disease (PD). Given that the only example of a protein that drives lysosomal Ca2+ import is a risk gene for PD, an understanding of its function, as well as identifying more regulators of lysosomal Ca2+ entry is of high significance. Catp-6 was identified as a potential lysosomal Ca2+ importer only because of a new fluorescent reporter called CalipHluor, with which one could quantitatively image lysosomal Ca2+. Here we propose to identify and catalog proteins that drive lysosomal Ca2+ import by screening nematodes that are engineered to show phenotypes at the whole worm, sub-cellular and lysosomal Ca2+ level. We have developed a system where, the deletion of a lysosomal Ca2+ importer will concurrently rescue lethality caused by the deletion of a lysosomal Ca2+ release channel, restore lysosome size and restore lysosomal Ca2+ - the latter revealed by Ca2+ mapping with CalipHluor. Since our methodology can map defects in lysosomal Ca2+ uptake, we propose to investigate two new molecular mechanisms that drive lysosomal Ca2+ import. The first is a gene we denote LCAX-1, that we identified from a preliminary screen in nematodes, that has putative Ca2+/H+ exchanger (CAX)-like function. While CAX genes have been described in diverse organisms, lysosomal CAX genes have eluded discovery in humans. We propose experiments that will test whether LCAX-1 behaves like a CAX at the lysosome and if so, it would constitute the first example of a human lysosomal CAX. The second corresponds to the Parkinson's risk gene ATP13A2 that has been previously described as an importer of various heavy metal ions. Yet our data suggest that it imports Ca2+. We propose experiments that will distinguish between these two possibilities and by mutational analysis we will identify the residues in ATP13A2 responsible for Ca2+ binding. Success in our aims will offer knowledge of new lysosomal Ca2+ import mechanisms, identify the elusive human lysosomal CAX gene, and provide structural insight into the functionality of a risk gene for Parkinson's disease e.g., identify the Ca2+ binding site in ATP13A2.

项目摘要溶酶体中许多已知的管腔钙离子调节器的缺陷导致明显的神经疾病通过扰乱溶酶体的钙离子水平。溶酶体Ca~(2+)可因任一种钙释放缺陷而失控渠道或传输者。虽然有几个溶酶体钙释放通道的例子，但也有只有一种促进溶酶体钙离子输入的蛋白质。这个单独的例子是通过以下方式确定的我的实验室是秀丽线虫的Catp-6。其人类同源基因ATP13A2是高血压的主要危险基因之一。帕金森氏病(PD)。考虑到驱动溶酶体钙离子输入的蛋白质的唯一例子是帕金森病的危险基因，了解其功能，以及识别更多的溶酶体钙调节因子进入具有很高的意义。由于一种新的荧光报告，CATP-6被确定为潜在的溶酶体钙离子导入者被称为CalipHluor，人们可以用它对溶酶体钙离子进行定量成像。在这里，我们建议确定并通过筛选线虫来分类驱动溶酶体钙离子输入的蛋白质全虫、亚细胞和溶酶体钙离子水平的表型。我们开发了一种系统，溶酶体Ca~(2+)导入蛋白的缺失将同时挽救因缺失溶酶体钙释放通道，恢复溶酶体大小和恢复溶酶体钙-后者揭示用CalipHluor定位Ca2+。由于我们的方法可以定位溶酶体钙摄取的缺陷，我们建议研究两个新的驱动溶酶体钙离子输入的分子机制。第一个是我们命名为LCAX-1的基因，我们从线虫的初步筛选中鉴定出，它具有可能的钙/氢交换(CAX)样功能。虽然CAX基因已经在不同的生物体中被描述，但溶酶体CAX基因还没有被发现在人类身上。我们提出的实验将测试LCAX-1在溶酶体上的行为是否像CAX 如果是这样的话，这将是人类溶酶体CAX的第一个例子。第二个对应于先前描述的帕金森病风险基因ATP13A2 作为各种重金属离子的进口商。然而，我们的数据表明，它进口了钙离子。我们建议将区分这两种可能性的实验，通过突变分析，我们将确定 ATP13A2上的残基负责与钙离子结合。我们的目标的成功将提供有关新的溶酶体钙离子输入机制的知识，识别难以捉摸的人溶酶体CAX基因，并提供了对风险基因功能的结构洞察例如，帕金森氏症，确定ATP13A2中的钙结合位点。