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）对于人类健康的许多方面（包括最佳免疫反应）是重要的微量营养素。 SE的生物学作用主要是通过将其掺入硒蛋白作为氨基酸硒代半胱氨酸（SEC）的生物作用。在人类中已经鉴定出25种硒蛋白，除一种外，所有甲蛋白蛋白也存在于小鼠和大鼠中的含SEC的蛋白质。尚未确定功能的一种硒蛋白是硒蛋白K（SEL K）。我们的初步数据表明，在免疫细胞中，SEL K蛋白表达最高，位于T细胞，单核细胞和巨噬细胞中的内质网（ER）膜。我们已经确定了SEL K和STIM1之间的新型相互作用，这是在免疫细胞激活过程中储存钙（Ca2+）进入的储存量所需的关键信号分子。此外，SEL K的表达降低会导致T细胞和巨噬细胞的Ca2+依赖性激活中的缺陷。这些发现导致了我们的中心假设，即SEL K通过将ER与质膜信号调节通过其与stim1和其他信号分子的相互作用，在CA2+依赖性激活中起关键作用。我们提出的研究目标是确定SEL K调节T细胞功能的特定机制，并阐明其在先天和适应性免疫中的总体作用。拟议的研究包括三个目标：特定目标1）。确定SEL K在T细胞激活期间调节从ER到质膜的信号传导的机制；特定目标2）。确定SEL K在免疫反应中的体内功能；和特定目标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细胞进行体外实验，以过表达SEL K的全长，突变或截短版本，以识别与STIM1相互作用所需的SEL K的域，并确定这些相互作用如何影响STIM1的sTIM1寡聚和下游CA2+依赖性依赖性依赖性TIP依赖性信号事件。此外，将使用共免疫沉淀和基于荧光的测定法确定SEL K和STIM1在T细胞激活过程中相互作用的程度。对于特定的目标2，我们开发了新型的转基因小鼠，其在T细胞（LCK-CRE）或包括巨噬细胞和中性粒细胞（Lyzs-cre）的SEL K（LCK-CRE）或髓样细胞中删除。这些小鼠将分析淋巴组织中T细胞和巨噬细胞的发育，周围组织中T细胞和巨噬细胞的激活和归纳，对抗原攻击的免疫反应以及EX Vivo T细胞和巨噬细胞的激活能力。对于特定的目标3，将使用三种菌株在T细胞（LCK-CRE），髓样细胞（LYZ-CRE）或脑神经元（CAMKII21-CRE）中删除的小鼠。已建立的方案将用于用WNV注射脚踏板感染这些小鼠。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荷，生存分析和其他读数来评估抗WNV免疫反应的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提供对SE影响免疫反应的机制的宝贵洞察力，并可能为与SE补充相比，可以为增强免疫细胞功能而具有更少的副作用提供更具选择性的治疗靶点。 公共卫生相关性：硒是一种必不可少的微量营养素，影响免疫力和硒的生物学作用主要是通过掺入硒蛋白中的。未鉴定功能的一种硒蛋白是硒蛋白K，我们发现它在免疫细胞中最丰富。该项目的目的是确定硒蛋白K如何参与调节免疫反应，并且该知识将为硒影响免疫反应的机制提供宝贵的见解。