Selenoprotein K modulates calcium-dependent signaling in immune cells

说明(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，包括最佳免疫反应。硒的生物学效应主要是通过其作为氨基酸--硒半胱氨酸(Sec)掺入硒蛋白中来实现的。已在人类中鉴定出25种硒蛋白，除了一种外，所有这些蛋白在小鼠和大鼠中也以含有SEC的蛋白的形式存在。一种功能尚未确定的硒蛋白是硒蛋白K(SelenoProtein 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。我们已经确定了Sel K和STIM1之间的一种新的相互作用，STIM1是在免疫细胞激活过程中存储操作钙进入所需的关键信号分子。此外，Sel K的表达减少导致T细胞和巨噬细胞的钙依赖激活功能缺陷。这些发现导致了我们的中心假设，即Sel K通过与STIM1和其他信号分子相互作用来调节ER到质膜的信号转导，在依赖钙的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，并阐明其在先天免疫和获得性免疫中的整体作用。建议的研究包括三个目标：具体目标1)。确定在T细胞激活过程中，Sel K调节从内质网到质膜的信号的机制；特定目标2)。确定Sel K在免疫反应中的体内功能；并针对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，以过表达全长、突变或截短版本的Sel K，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寡聚和T细胞激活过程中下游依赖钙的信号事件。此外，Sel K和STIM1在T细胞激活过程中的相互作用程度将通过免疫共沉淀和基于荧光的分析来确定。针对特定目的2，我们开发了T细胞(LCK-CRE)或包括巨噬细胞和中性粒细胞在内的髓系细胞(Lyzs-CRE)中缺失Sel K的新型转基因小鼠。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，对抗原攻击的免疫反应，以及体外T细胞和巨噬细胞的激活能力。对于特定的目的3，将使用T细胞(LCK-CRE)、髓细胞(Lyzs-CRE)或脑神经元(CaMKII21-CRE)中Sel K缺失的三个品系的小鼠。一项既定的方案将被用来用脚垫注射西尼罗河病毒感染这些小鼠。然后，我们将通过测量血浆抗WNV IgM、外周组织和脑中的病毒载量、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有助于深入了解Se影响免疫反应的机制，并可能为增强免疫细胞功能提供一个比补充Se更少副作用的更具选择性的治疗靶点。 与公共健康相关：硒是一种影响免疫的基本微量营养素，其生物学效应主要是通过将其掺入硒蛋白来发挥的。一种功能尚未确定的硒蛋白是硒蛋白K，我们发现它在免疫细胞中含量最高。这个项目的目标是确定硒蛋白K是如何参与调节免疫反应的，这一知识将为研究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.