Selenoprotein K modulates calcium-dependent signaling in immune cells

描述（由申请人提供）：硒（Se）是一种对人类健康的许多方面都很重要的必需微量营养素，包括最佳免疫反应。硒的生物学效应主要是通过其作为氨基酸硒代半胱氨酸（Sec）掺入硒蛋白而发挥的。在人类中已经鉴定出25种硒蛋白，其中除了一种之外，所有的硒蛋白也以含Sec蛋白的形式存在于小鼠和大鼠中。硒蛋白K（Selenoprotein K，Sel K）是一种功能尚未确定的硒蛋白。我们的初步数据表明，Sel K蛋白表达在免疫细胞中最高，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STIM 1之间的一种新的相互作用，STIM 1是免疫细胞激活期间钙（Ca 2+）进入所需的储存操作的关键信号分子。此外，减少表达的Sel K导致缺陷的Ca 2+依赖性激活的T细胞和巨噬细胞。这些发现导致了我们的中心假设，即Sel K通过与STIM 1和其他信号分子的相互作用调节ER至质膜信号传导，在免疫细胞的Ca 2+依赖性活化中起关键作用。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，并阐明其在先天性和适应性免疫中的整体作用。该研究包括三个目标：具体目标1）。确定在T细胞活化期间Sel K调节从ER到质膜的信号传导的机制;特异性目的2）。确定Sel K在免疫应答中的体内功能;和特异性目的3）。确定Sel K在防止病毒感染中的作用。我们的实验设计涉及使用HEK 293和Jurkat T细胞进行的体外实验，用于过表达全长、突变或截短形式的Sel K，以鉴定与STIM 1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STIM 1寡聚化和T细胞活化期间的下游Ca 2+依赖性信号传导事件。此外，将使用免疫共沉淀和基于荧光的测定来确定Sel K和STIM 1在T细胞活化过程中相互作用的程度。对于特定目标2，我们已经开发了在T细胞（Lck-Cre）或骨髓细胞（包括巨噬细胞和嗜中性粒细胞（Lyzs-Cre））中缺失Sel K的新型转基因小鼠。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细胞和巨噬细胞的活化和归巢、对抗原激发的免疫应答以及离体T细胞和巨噬细胞的活化能力。对于特异性目标3，将使用在T细胞（Lck-Cre）、髓样细胞（Lyzs-Cre）或脑神经元（CaMK II 21-Cre）中缺失Sel K的三种小鼠品系。将利用已建立的方案通过脚垫注射WNV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免疫应答水平和感染严重程度。总的来说，阐明Sel K在免疫细胞中的功能将为Se影响免疫应答的机制提供有价值的见解，并可能提供更具选择性的治疗靶点，用于增强免疫细胞功能，与Se补充剂相比副作用更少。 公共卫生相关性：硒是一种影响免疫力的必需微量营养素，硒的生物学效应主要通过硒蛋白发挥。一种没有功能的硒蛋白是硒蛋白K，我们发现它在免疫细胞中最丰富。该项目的目标是确定硒蛋白K如何参与调节免疫反应，这些知识将为硒影响免疫反应的机制提供有价值的见解。

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.