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Calcium homeostasis in organelles

细胞器中的钙稳态

基本信息

批准号：
10631101
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/10631101
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 Human Image Impairment Ions Knowledge Laboratories Lead Lysosomes Maps Measures Mediating Metal Ion Binding Metals Methodology Modeling Molecular Mutation Analysis Nematoda Neurodegenerative Disorders Normalcy Organelles Organism PARK9 gene Parkinson Disease Pathway interactions Phenotype Proteins Reporter Reporting Saccharomyces cerevisiae Swelling System Testing Variant Whole Organism 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.

项目摘要许多已知的溶酶体内腔Ca 2+调节因子的缺陷导致不同的神经系统疾病通过破坏溶酶体钙离子水平。溶酶体Ca 2+可通过Ca 2+释放缺陷而失调通道或传输器。虽然存在溶酶体Ca 2+释放通道的几个实例，但存在这仅仅是促进溶酶体Ca 2+输入的蛋白质的一个例子。这一独特的例子被发现，我的实验室是C区的catp-6优美的它的人类同源物ATP 13 A2是一种主要的风险基因，帕金森病（PD）。考虑到驱动溶酶体Ca 2+输入的蛋白质的唯一例子是 PD的风险基因，了解其功能，以及确定更多的溶酶体Ca 2+调节剂进入具有重要意义。 Catp-6被鉴定为潜在的溶酶体Ca 2+输入者，仅仅是因为一个新的荧光报告基因称为CalipHluor，可以定量成像溶酶体Ca 2+。在这里，我们建议确定并通过筛选线虫来分类驱动溶酶体Ca 2+输入的蛋白质，在整个蠕虫，亚细胞和溶酶体Ca 2+水平的表型。我们开发了一个系统，溶酶体Ca 2+输入者的缺失将同时挽救由溶酶体Ca 2+输入者的缺失引起的致死性。溶酶体Ca 2+释放通道，恢复溶酶体大小和恢复溶酶体Ca 2 + -后者显示，用CalipHluor绘制Ca 2+图。由于我们的方法可以映射溶酶体Ca 2+摄取的缺陷，我们建议研究两个新的驱动溶酶体Ca 2+输入的分子机制。第一个基因我们用LCAX-1表示，从线虫的初步筛选中鉴定，其具有推定的Ca 2 +/H+交换器（CAX）样功能。虽然CAX基因已在多种生物体中被描述，但溶酶体CAX基因尚未被发现在人类身上。我们提出实验来测试LCAX-1在溶酶体中的行为是否像CAX 如果是这样的话，它将构成人类溶酶体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ATP 13 A2 作为各种重金属离子的进口商。然而，我们的数据表明，它输入Ca 2+。我们提出实验将区分这两种可能性，并通过突变分析，我们将确定 ATP 13 A2中负责Ca 2+结合的残基。我们目标的成功将提供新的溶酶体Ca 2+输入机制的知识，识别难以捉摸的人溶酶体CAX基因，并提供结构洞察的风险基因的功能，帕金森氏病，例如，确定ATP 13 A2中的Ca 2+结合位点。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Passive endocytosis in model protocells.

DOI：
10.1073/pnas.2221064120
发表时间：
2023-06-13
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Zhang, Stephanie J.;Lowe, Lauren A.;Anees, Palapuravan;Krishnan, Yamuna;Fai, Thomas G.;Szostak, Jack W.;Wang, Anna
通讯作者：
Wang, Anna

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.

DOI：
10.1073/pnas.2113174118
发表时间：
2021-10-12
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Suresh B;Saminathan A;Chakraborty K;Zajac M;Cui C;Becker L;Krishnan Y
通讯作者：
Krishnan Y

Tissue-specific targeting of DNA nanodevices in a multicellular living organism.

DOI：
10.7554/elife.67830
发表时间：
2021-07-28
期刊：
eLife
影响因子：
7.7
作者：
Chakraborty K;Anees P;Surana S;Martin S;Aburas J;Moutel S;Perez F;Koushika SP;Kratsios P;Krishnan Y
通讯作者：
Krishnan Y

The evolution of organellar calcium mapping technologies.