Selenoprotein K modulates calcium-dependent signaling in immune cells

描述（由申请人提供）：硒（Se）是一种必需的微量营养素，对人体健康的许多方面都很重要，包括最佳免疫反应。硒的生物学效应主要通过硒作为氨基酸硒半胱氨酸（Sec）结合到硒蛋白中发挥作用。在人体中已经鉴定出25种硒蛋白，其中除一种外，其余都以含硒蛋白的形式存在于小鼠和大鼠体内。一种没有功能的硒蛋白是硒蛋白K （selk）。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。我们已经确定了Sel K和STIM1之间的一种新的相互作用，STIM1是免疫细胞激活期间钙（Ca2+）进入所需的储存操作的关键信号分子。此外，Sel K的表达减少导致T细胞和巨噬细胞Ca2+依赖性激活的缺陷。这些发现导致了我们的中心假设，即Sel K通过与STIM1和其他信号分子的相互作用调节ER到质膜信号传导，在免疫细胞的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细胞功能的具体机制，并阐明其在先天免疫和适应性免疫中的整体作用。提出的研究包括三个目标：具体目标1)。确定selk在T细胞活化过程中调控内质网到质膜信号的机制；具体目标2)。测定体内Sel K在免疫应答中的功能；和具体目标3)。确定selk在预防病毒感染中的作用。我们的实验设计包括使用HEK293和Jurkat T细胞进行过表达全长，突变或截断版本的Sel K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寡聚化和下游Ca2+依赖性信号事件在T细胞活化过程中。此外，在T细胞活化过程中，Sel K和STIM1相互作用的程度将通过免疫共沉淀和基于荧光的检测来确定。对于Specific Aim 2，我们开发了一种新的转基因小鼠，在T细胞（Lck-Cre）或骨髓细胞（包括巨噬细胞和中性粒细胞）中缺失Sel K （lyz - cre）。这些小鼠将被分析淋巴组织中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、对抗原挑战的免疫反应以及离体T细胞和巨噬细胞的激活能力。对于特异性Aim 3，将使用三种小鼠品系，它们在T细胞（Lck-Cre）、髓细胞（lyz - cre）或脑神经元（CaMKII21-Cre）中缺失Sel K。将采用既定方案，在这些小鼠足部注射西尼罗河病毒感染。然后，我们将通过测量血浆抗西尼罗河病毒IgM、外周组织和大脑中的病毒载量、生存分析和其他读数，以时间过程的方式评估抗西尼罗河病毒免疫反应水平和感染严重程度。总之，阐明硒K在免疫细胞中的功能将为硒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，与硒补充相比，副作用更少。

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.