Selenoprotein K modulates calcium-dependent signaling in immune cells

硒蛋白 K 调节免疫细胞中的钙依赖性信号传导

• 批准号: 8492019
• 负责人: Peter R Hoffmann
• 金额: $ 34.9万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8492019
• 关键词: Adverse effects; Affect; Amino Acids; Biological; Biological Assay; Biology; Blood - brain barrier anatomy; Brain; Calcium; Cell membrane; Cell physiology; Cells; Co-Immunoprecipitations; Data; Defect; Encephalitis; Endoplasmic Reticulum; Ensure; Event; Experimental Designs; Fluorescence; Goals; Health; Homing; Human; Immune; Immune Cell Activation; Immune response; Immunity; Immunoglobulin M; Immunologist; In Vitro; Infection; Injection of therapeutic agent; Knowledge; Length; Lymphoid Tissue; Measures; Membrane; Micronutrients; Modeling; Mouse Strains; Mus; Mutate; Myeloid Cells; Natural Immunity; Neurons; Perinatal; Peripheral; Plasma; Play; Predisposition; Principal Investigator; Proteins; Protocols documentation; Rattus; Research; Role; STIM1 gene; Scaffolding Protein; Selenium; Selenocysteine; Severities; Signal Transduction; Signaling Molecule; Supplementation; Survival Analysis; T-Cell Activation; T-Cell Development; T-Lymphocyte; Time; Tissues; Training; Transgenic Mice; Viral Load result; Virus Diseases; West Nile virus; Wild Type Mouse; adaptive immunity; base; brain tissue; immune function; in vivo; insight; macrophage; monocyte; mouse model; neutrophil; novel; overexpression; pathogen; protein expression; public health relevance; research study; selenoprotein; therapeutic target

DESCRIPTION (provided by applicant): Selenium (Se) is an essential micronutrient important for many aspects of human health, including optimal immune responses. The biological effects of Se are exerted mainly through its incorporation into selenoproteins as the amino acid, selenocysteine (Sec). Twenty-five selenoproteins have been identified in humans, all but one of which also exists as Sec-containing proteins in mice and rats. One selenoprotein for which no function has been identified is selenoprotein K (Sel K). Our preliminary data indicate Sel K protein expression is highest in immune cells, localizing to the endoplasmic reticulum (ER) membrane in T cells, monocytes, and macrophages. We have identified a novel interaction between Sel K and STIM1, which is a key signaling molecule required for store-operated for calcium (Ca2+) entry during activation of immune cells. Furthermore, reduced expression of Sel K caused defects in Ca2+-dependent activation of T cells and macrophages. These findings have led to our central hypothesis that Sel K plays a key role in Ca2+-dependent activation of immune cells by regulating ER to plasma membrane signaling through its interactions with STIM1 and other signaling molecules. Our proposed research objective is to determine specific mechanisms by which Sel K regulates the functions of T cells and elucidate its overall role in innate and adaptive immunity. The proposed study includes three aims: Specific Aim 1). Determine the mechanisms by which Sel K regulates signaling from ER to the plasma membrane during the activation of T cells; Specific Aim 2). Determine the in vivo function of Sel K in immune responses; and Specific Aim 3). Determine the role of Sel K in protecting against viral infection. Our experimental design involves in vitro experiments using HEK293 and Jurkat T cells for overexpression of full-length, mutated, or truncated versions of Sel K to identify domains of Sel K required for interactions with STIM1, and to determine how these interactions affect STIM1 oligomerization and downstream Ca2+-dependent signaling events during T cell activation. In addition, the extent to which Sel K and STIM1 interact over the course of T cell activation will be determined using co-immunoprecipitation and fluorescence-based assays. For Specific Aim 2, we have developed novel transgenic mice with Sel K deleted in T cells (Lck-Cre) or myeloid cells including macrophages and neutrophils (Lyzs-Cre). These mice will be analyzed for development of T cells and macrophages in lymphoid tissues, activation and homing of T cell and macrophage in peripheral tissues, immune responses to antigenic challenge, and activation capacity of ex vivo T cells and macrophages. For Specific Aim 3, three strains of mice will be used that have Sel K deleted in T cells (Lck-Cre), myeloid cells (Lyzs-Cre), or brain neurons (CaMKII21-Cre). An established protocol will be utilized to infect these mice with footpad injections of WNV. We will then evaluate levels of anti-WNV immune responses and severity of infection in a time-course manner by measuring plasma anti-WNV IgM, viral load in peripheral tissues and brain, survival analyses, and other readouts. Overall, elucidating the function of Sel K in immune cells will provide valuable insight into mechanisms by which Se influences immune responses and may provide a more selective therapeutic target for augmenting immune cell function with fewer side-effects compared to Se supplementation. PUBLIC HEALTH RELEVANCE: Selenium is an essential micronutrient that influences immunity and the biological effects of selenium are exerted mainly through its incorporation into selenoproteins. One selenoprotein for which no function has been identified is selenoprotein K, which we have found is most abundant in immune cells. The goal of this project is to determine how selenoprotein K is involved in modulating immune responses and this knowledge will provide valuable insight into mechanisms by which selenium influences immune responses.

描述（由申请人提供）：硒 (Se) 是一种必需的微量营养素，对人类健康的许多方面都很重要，包括最佳的免疫反应。 Se 的生物效应主要通过其以氨基酸硒代半胱氨酸 (Sec) 的形式掺入硒蛋白中来发挥。已在人类中鉴定出 25 种硒蛋白，除其中一种外，所有这些蛋白在小鼠和大鼠中也以含 Sec 的蛋白质形式存在。一种尚未确定其功能的硒蛋白是硒蛋白 K (Sel K)。我们的初步数据表明 Sel K 蛋白在免疫细胞中表达最高，定位于 T 细胞、单核细胞和巨噬细胞的内质网 (ER) 膜。我们发现了 Sel K 和 STIM1 之间的一种新型相互作用，STIM1 是免疫细胞激活过程中钙 (Ca2+) 进入库操作所需的关键信号分子。此外，Sel K 表达减少会导致 T 细胞和巨噬细胞的 Ca2+ 依赖性激活缺陷。这些发现引出了我们的中心假设：Sel K 通过与 STIM1 和其他信号分子的相互作用来调节 ER 至质膜信号传导，从而在 Ca2+ 依赖性免疫细胞激活中发挥关键作用。我们提出的研究目标是确定 Sel K 调节 T 细胞功能的具体机制，并阐明其在先天性和适应性免疫中的总体作用。拟议的研究包括三个目标： 具体目标 1)。确定 Sel K 在 T 细胞激活过程中调节 ER 至质膜信号传导的机制；具体目标2)。确定 Sel K 在免疫反应中的体内功能；和具体目标 3)。确定 Sel K 在预防病毒感染中的作用。我们的实验设计涉及使用 HEK293 和 Jurkat T 细胞过度表达 Sel K 全长、突变或截短版本的体外实验，以确定与 STIM1 相互作用所需的 Sel K 结构域，并确定这些相互作用如何影响 STIM1 寡聚化和T 细胞激活过程中下游 Ca2+ 依赖性信号传导事件。此外，Sel K 和 STIM1 在 T 细胞激活过程中相互作用的程度将使用免疫共沉淀和基于荧光的测定来确定。对于特定目标 2，我们开发了新型转基因小鼠，其中 T 细胞 (Lck-Cre) 或包括巨噬细胞和中性粒细胞 (Lyzs-Cre) 在内的骨髓细胞中删除了 Sel K。将分析这些小鼠淋巴组织中 T 细胞和巨噬细胞的发育、外周组织中 T 细胞和巨噬细胞的激活和归巢、对抗原攻击的免疫反应以及离体 T 细胞和巨噬细胞的激活能力。对于特定目标 3，将使用 T 细胞 (Lck-Cre)、骨髓细胞 (Lyzs-Cre) 或脑神经元 (CaMKII21-Cre) 中 Sel K 缺失的三种小鼠品系。将利用既定方案通过足垫注射西尼罗河病毒来感染这些小鼠。然后，我们将通过测量血浆抗 WNV IgM、外周组织和大脑中的病毒载量、生存分析和其他读数，以时间进程的方式评估抗 WNV 免疫反应水平和感染的严重程度。总体而言，阐明 Sel K 在免疫细胞中的功能将为了解 Se 影响免疫反应的机制提供有价值的见解，并可能为增强免疫细胞功能提供更具选择性的治疗靶点，并且与补充 Se 相比，副作用更少。 公共健康相关性：硒是一种影响免疫力的必需微量营养素，硒的生物效应主要通过其与硒蛋白的结合来发挥。硒蛋白 K 是一种尚未确定其功能的硒蛋白，我们发现它在免疫细胞中含量最多。该项目的目标是确定硒蛋白 K 如何参与调节免疫反应，这些知识将为了解硒影响免疫反应的机制提供有价值的见解。