The Regulation of Orai1 and STIM1 by Dynamic S-Acylation

动态 S-酰化对 Orai1 和 STIM1 的调节

• 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
• 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

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.

摘要 钙库操作的钙进入是适当的T细胞活化和发育的必要过程。两 促进这一过程的蛋白质是Orai 1和STIM 1。Orai 1定位于质膜（PM）。 STIM 1定位于内质网（ER）膜，并在其内具有钙敏感EF手 ER腔。在T细胞受体激活后，ER钙储存被耗尽，激活STIM 1，然后 经历伸长构象变化，并在ER-PM处与Orai 1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是 然而，对于适当的T细胞信号传导至关重要的是，调节定位的潜在分子机制 Orai 1和STIM 1与ER-PM连接点的关系尚不清楚。我们发现Orai 1和STIM 1都是S- 酰化的S-酰化是半胱氨酸残基被DHHC蛋白家族翻译后脂化 酰基转移酶，并已发现调节蛋白质定位和运输。因此本项目 目的是确定Orai 1和STIM 1的S-酰化对适当定位和钙通道功能的作用， 并确定负责S-酰化的DHHC酶。S-酰化的重要作用 Orai 1/STIM 1将使用生物化学和先进的活细胞成像方法进行评价。这些 互补技术将确定Orai 1和STIM 1的S-酰化对ER定位的重要性。 PM连接、钙库操作的钙进入和下游T细胞信号传导。这将在两个方面进行研究。 具体目标。在目标1中，我们将确定Orai 1和STIM 1 S-酰化对复合物的要求 形成和储存操作的钙进入。利用高分辨率显微镜技术和功能 我们将检验快速和瞬时T细胞受体诱导的Orai 1的S-酰化和 STIM 1对于Orai 1/STIM 1复合物的形成和下游商店操作的钙进入至关重要。在目标2中， 我们将确定哪些酶介导Orai 1和STIM 1的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。在鉴定出负责 Orai 1/STIM 1 S-酰化，我们将使用生物化学方法来确定酶对T细胞的影响， 增殖和分化。由于Orai 1和STIM 1中的功能获得性和功能丧失性突变 导致疾病，如Störmorken综合征和严重的联合免疫缺陷，这项研究还将 描述了潜在的新的治疗靶点。