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.

项目概要溶酶体中许多已知的管腔 Ca2+ 调节因子的缺陷会导致不同的神经系统疾病通过破坏溶酶体 Ca2+ 水平。溶酶体 Ca2+ 可能因 Ca2+ 释放缺陷而失调渠道或运输商。虽然有几个溶酶体 Ca2+ 释放通道的例子，但这只是促进溶酶体 Ca2+ 输入的蛋白质的一个例子。这个孤立的例子是由我的实验室是在线虫中的 catp-6。其人类同源物 ATP13A2 是主要风险基因之一帕金森病（PD）。鉴于驱动溶酶体 Ca2+ 输入的蛋白质的唯一例子是 PD 风险基因，了解其功能，以及识别更多溶酶体 Ca2+ 调节因子入境意义重大。仅由于新的荧光报告基因，Catp-6 才被确定为潜在的溶酶体 Ca2+ 输入者称为 CalipHluor，可以对溶酶体 Ca2+ 进行定量成像。在这里我们建议识别并通过筛选经过工程改造的线虫来对驱动溶酶体 Ca2+ 输入的蛋白质进行分类整个蠕虫、亚细胞和溶酶体 Ca2+ 水平的表型。我们开发了一个系统，删除溶酶体 Ca2+ 输入蛋白将同时挽救因删除溶酶体 Ca2+ 释放通道，恢复溶酶体大小并恢复溶酶体 Ca2+ - 后者揭示使用 CalipHluor 进行 Ca2+ 映射。由于我们的方法可以绘制溶酶体 Ca2+ 吸收的缺陷，因此我们建议研究两种新的驱动溶酶体 Ca2+ 输入的分子机制。第一个是我们表示为 LCAX-1 的基因，我们通过线虫的初步筛选鉴定出，其具有假定的 Ca2+/H+ 交换器 (CAX) 样功能。虽然 CAX 基因已在多种生物体中得到描述，但溶酶体 CAX 基因尚未被发现在人类中。我们提出了一些实验来测试 LCAX-1 在溶酶体中是否表现得像 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输入 Ca2+。我们建议区分这两种可能性的实验，通过突变分析，我们将确定 ATP13A2 中负责 Ca2+ 结合的残基。我们目标的成功将提供新的溶酶体 Ca2+ 输入机制的知识，识别难以捉摸的人溶酶体 CAX 基因，并为风险基因的功能提供结构见解例如帕金森病，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）

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

管状溶酶体具有活性离子梯度和平衡巨噬细胞的吞噬作用。

DOI：
发表时间：
2021
期刊：
影响因子：
11.1
作者：
Suresh, Bhavyashree;Saminathan, Anand;Chakraborty, Kasturi;Zajac, Matthew;Cui, Chang;Becker, Lev;Krishnan, Yamuna
通讯作者：
Krishnan, Yamuna

Detecting organelle-specific activity of potassium channels with a DNA nanodevice.

使用 DNA 纳米装置检测钾通道的细胞器特异性活性。

DOI：
发表时间：
2023-09-21
期刊：
影响因子：
46.9
作者：
Anees, Palapuravan;Saminathan, Anand;Rozmus, Ezekiel R;Di, Anke;Malik, Asrar B;Delisle, Brian P;Krishnan, Yamuna
通讯作者：
Krishnan, Yamuna

A DNA nanodevice for mapping sodium at single-organelle resolution.

一种用于以单细胞器分辨率绘制钠图谱的 DNA 纳米装置。

DOI：
10.1038/s41587-023-01950-1
发表时间：
2023-09-21
期刊：
Nature biotechnology
影响因子：
46.9
作者：
Junyi Zou;Koushambi Mitra;Palapuravan Anees;Daphne Oettinger;Joseph R Ramirez;Aneesh T. Veetil;Priyanka Dutta Gupta;Rajini Rao;Jayson J. Smith;Paschalis Kratsios;Yamuna Krishnan
通讯作者：
Yamuna Krishnan

Passive endocytosis in model protocells.

模型原始细胞中的被动内吞作用。

DOI：
发表时间：
2023-06-13
期刊：
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

The Cell: A Molecular Approach

细胞：分子方法