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.

项目摘要溶酶体中许多已知的Lumenal Ca2+调节剂缺陷导致独特的神经系统疾病通过破坏溶酶体Ca2+水平。溶酶体Ca2+可能因任何Ca2+释放中的缺陷而失调通道或转运蛋白。虽然有几个溶酶体CA2+释放通道的示例，但有只有一个促进溶酶体Ca2+进口的蛋白质的例子。这个孤独的例子由我的实验室是秀丽隐杆线虫中的Catp-6。它的人类同源物ATP13A2是主要风险基因之一帕金森氏病（PD）。鉴于驱动溶酶体Ca2+进口的蛋白质的唯一例子是 PD的风险基因，对其功能的理解，并确定更多的溶酶体CA2+调节剂进入具有很高的意义。 CATP-6仅由于新的荧光报告基因而被鉴定为潜在的溶酶体Ca2+进口商称为caliphluor，可以用它定量图像溶酶体Ca2+。在这里我们建议确定通过筛选用于显示的线虫来驱动溶酶体Ca2+进口的目录蛋白整个蠕虫，亚细胞和溶酶体Ca2+水平的表型。我们已经开发了一个系统，溶酶体Ca2+进口商的删除将同时挽救因删除而引起的致命性溶酶体Ca2+释放通道，恢复溶酶体大小和恢复溶酶体Ca2+ - 后者由 Ca2+用caliphluor映射。由于我们的方法可以在溶酶体Ca2+摄取中绘制缺陷，因此我们建议研究两个新的驱动溶酶体Ca2+进口的分子机制。第一个是我们表示lcax-1的基因，我们从线虫中的初步屏幕中识别，该屏幕具有推定的Ca2+/H+交换器（CAX）类似功能。虽然已经在各种生物体中描述了CAX基因，但溶酶体CAX基因已避免了发现在人类中。我们提出的实验将测试LCAX-1是否在溶酶体处表现得像CAX 如果是这样，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，我们的数据表明它导入CA2+。我们建议可以区分这两种可能性和突变分析的实验，我们将确定 ATP13A2中的残基负责Ca2+结合。我们的目标成功将提供有关新溶酶体CA2+进口机制的知识，请确定难以捉摸的人类溶酶体CAX基因，并提供有关风险基因功能的结构洞察力帕金森氏病，例如，识别ATP13A2中的Ca2+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

The evolution of organellar calcium mapping technologies.

DOI：
10.1016/j.ceca.2022.102658
发表时间：
2022-12
期刊：
CELL CALCIUM
影响因子：
4
作者：
Zajac, Matthew;Modi, Souvik;Krishnan, Yamuna
通讯作者：
Krishnan, Yamuna

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