The Regulation of Orai1 and STIM1 by Dynamic S-Acylation

动态 S-酰化对 Orai1 和 STIM1 的调节

基本信息

批准号：
10560518
负责人：
Savannah Joy West Diaz
金额：
$ 1.45万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10560518
关键词：
Acylation Binding Biochemical Biological Assay Calcium Calcium Channel Calcium-Binding Domain Cell Differentiation process Cell membrane Cell physiology Cells Complex Cysteine Data Development Disease EF Hand Motifs Endoplasmic Reticulum Enzymes Ion Channel Mediating Mediator Membrane Microscopy Molecular Molecular Conformation Mutagenesis Play Post-Translational Protein Processing Process Protein Family Proteins Proteome Proteomics Receptor Activation Receptor Signaling Regulation Research Resolution Role STIM1 gene Severe Combined Immunodeficiency Signal Pathway Signal Transduction Syndrome T-Cell Activation T-Cell Proliferation T-Cell Receptor T-Lymphocyte Techniques Testing Tubular Aggregate Myopathies ZAP-70 Gene congenital immunodeficiency disease-causing mutation effector T cell experimental study gain of function gain of function mutation imaging approach immunological synapse in vivo knock-down live cell imaging loss of function loss of function mutation new therapeutic target novel protein acyltransferase receptor recruit small hairpin RNA therapeutic target thioester trafficking

项目摘要

Abstract Store operated calcium entry is an essential process for proper T cell activation and development. The two proteins that facilitate this process are Orai1 and STIM1. Orai1 is localized to the plasma membrane (PM). STIM1 is localized to the endoplasmic reticulum (ER) membrane, and has a calcium sensing EF hand within the ER lumen. Upon T cell receptor activation, ER calcium stores are depleted, activating STIM1, which then undergoes an elongating conformational change, and binds to and forms complexes with Orai1 at ER-PM junctions, resulting in calcium entry into the cell. The formation of these complexes has been shown to be critical for proper T cell signaling, however, the underlying molecular mechanisms that regulate the localization of Orai1 and STIM1 to ER-PM junctions are still unknown. We have found that both Orai1 and STIM1 are S- acylated. S-acylation is the post-translational lipidation of cysteine residues by the DHHC family of protein acyltransferases, and has been found to regulate protein localization and trafficking. Therefore, this project aims to define the role of S-acylation of Orai1 and STIM1 on proper localization and calcium channel function, and determine the DHHC enzymes responsible for S-acylation. The essential role of S-acylation of Orai1/STIM1 will be evaluated using biochemical and advanced live cell imaging approaches. These complementary techniques will define the importance of S-acylation of Orai1 and STIM1 on localization to ER- PM junctions, store operated calcium entry, and downstream T cell signaling. This will be studied in two specific aims. In Aim 1, we will determine the requirement of Orai1 and STIM1 S-acylation on complex formation and store operated calcium entry. Utilizing high resolution microscopy techniques and functional assays, we will test the hypothesis that rapid and transient T cell receptor-induced S-acylation of Orai1 and STIM1 is essential for Orai1/STIM1 complex formation and downstream store operated calcium entry. In Aim 2, we will establish what enzymes mediate Orai1 and STIM1 S-acylation and evaluate their effects on downstream T cell receptor signaling and effector function. Upon identification of the enzymes responsible for Orai1/STIM1 S-acylation, we will use biochemical approaches to determine the effect of the enzymes on T cell proliferation and differentiation. As both gain-of-function and loss-of-function mutations in Orai1 and STIM1 cause diseases such as Störmorken syndrome and severe combined immunodeficiency, this research will also describe potential novel therapeutic targets.

抽象的钙库操作的钙进入是 T 细胞正常激活和发育的重要过程。促进这一过程的蛋白质是 Orai1，而 STIM1 位于质膜 (PM)。 STIM1 定位于内质网 (ER) 膜，内有钙感应 EF 手 T 细胞受体激活后，ER 腔内的钙储备就会耗尽，从而激活 STIM1，然后 STIM1 就会被激活。经历伸长的构象变化，并在 ER-PM 处与 Orai1 结合并形成复合物连接，导致钙进入细胞。这些复合物的形成已被证明。然而，对于正确的 T 细胞信号传导至关重要，调节定位的潜在分子机制 Orai1 和 STIM1 与 ER-PM 连接的关系仍然未知。我们发现 Orai1 和 STIM1 都是 S-的。 S-酰化是 DHHC 蛋白家族对半胱氨酸残基的翻译后脂化。酰基转移酶，并已被发现可以调节蛋白质定位和运输，因此，该项目。旨在确定 Orai1 和 STIM1 的 S-酰化对正确定位和钙通道功能的作用，并确定负责S-酰化的DHHC酶对S-酰化的重要作用。 Orai1/STIM1 将使用生化和先进的活细胞成像方法进行评估。补充技术将定义 Orai1 和 STIM1 的 S-酰化对 ER- 定位的重要性 PM 连接、钙池操作的钙进入和下游 T 细胞信号传导将分两部分进行研究。在目标1中，我们将确定Orai1和STIM1 S-酰化对复合物的要求。利用高分辨率显微镜技术和功能进行形成和储存操作。分析中，我们将测试以下假设：快速且短暂的 T 细胞受体诱导的 Orai1 和 STIM1 对于 Orai1/STIM1 复合物的形成和下游存储操作的钙进入至关重要。我们将确定哪些酶介导 Orai1 和 STIM1 S-酰化并评估它们对下游 T 细胞受体信号传导和效应器功能的鉴定。 Orai1/STIM1 S-酰化，我们将使用生化方法来确定酶对 T 细胞的影响 Orai1 和 STIM1 的功能获得和功能丧失突变。导致 Störmorken 综合征和严重联合免疫缺陷等疾病，这项研究还将描述潜在的新治疗靶点。