Role of Immunoproteasomes in Activated T Cell Apoptosis

描述（由申请人提供）：T细胞响应同源抗原的克隆扩增最终导致大多数扩增的T细胞通过程序性细胞死亡（细胞凋亡）而缺失，只有少数存活下来成为记忆细胞。这个过程对于在急性免疫应答终止后恢复T细胞稳态至关重要，这促进了保护性免疫并阻止了自身免疫。虽然现在有强有力的证据表明Bcl-2家族的蛋白质，特别是抗凋亡Bcl-2和促凋亡Bim，在介导体内活化的T细胞凋亡中起重要作用，但对这些蛋白质本身如何控制知之甚少。调节Bcl-2家族成员的一种机制是泛素/蛋白酶体介导的降解。T细胞具有称为“免疫蛋白酶体”的特化蛋白酶体，其含有三种干扰素-g-诱导的催化免疫亚基：LMP 2、LMP 7和MECL-1。我们最近观察到，来自缺乏MECL-1和LMP 7的免疫蛋白酶体缺陷小鼠的T细胞在体内被超抗原激活后表现出过度的凋亡。相反，正常超抗原活化T细胞中LMP 7的过表达显著抑制细胞凋亡。总之，这些结果表明，免疫蛋白酶体在调节活化T细胞凋亡的幅度中发挥作用。鉴于Bcl-2家族成员在介导活化T细胞凋亡中的核心作用，以及几个Bcl-2家族成员是蛋白酶体底物，我们假设免疫蛋白酶体通过降解Bcl-2家族成员调节活化T细胞的Bim驱动的凋亡的幅度。我们将在以下具体目标调查这一假设：1。确定免疫蛋白酶体在促凋亡Bcl-2家族成员的降解和功能中的作用。该目的将涉及三重敲除小鼠（Bim-/-/MECL- 1-/-/LMP 7-/-）的产生，以确定免疫蛋白酶体缺乏的促凋亡作用是否需要Bim，并且该目的还将评估免疫蛋白酶体缺乏对活化的T细胞中促凋亡Bcl-2家族成员的功能和降解的影响。2.识别免疫蛋白酶体的结构和功能特征，这些特征对它们的抗凋亡活性至关重要。这一目标将涉及测试LMP 7和相关蛋白酶体亚基的活性位点和组装突变体调节活化T细胞凋亡的能力，并确定正常活化T细胞中的免疫蛋白酶体表达是否与凋亡活性相关。从长远来看，阐明免疫蛋白酶体在活化的T细胞凋亡中的作用的分子机制将进一步加深我们对免疫记忆发展的理解，并可能确定用于增强参与自身免疫性疾病发病机制的自身反应性T细胞凋亡或抑制T细胞凋亡的靶点，这些靶点可能有助于控制感染性或恶性疾病。对“威胁”（如感染或肿瘤）的免疫反应必须以这样一种方式控制，即提供记忆以防止未来类似的威胁（免疫），同时防止对自身的不必要的伤害（自身免疫）。该项目的目标是提高我们对参与该控制系统的分子的理解，这可能导致更好地治疗该系统自我破坏或崩溃的疾病（感染，癌症或自身免疫性疾病）。

DESCRIPTION (provided by applicant): Clonal expansion of T cells in response to cognate antigen eventually leads to deletion of most of the expanded T cells via programmed cell death (apoptosis), with only a minority surviving to become memory cells. This process is crucial for restoring T cell homeostasis after termination of an acute immune response, which promotes protective immunity and deters autoimmunity. While there is now strong evidence that proteins of the Bcl-2 family, particularly anti-apoptotic Bcl-2 and pro-apoptotic Bim, play an important role in mediating apoptosis of T cells activated in vivo, little is known about how these proteins themselves are controlled. One mechanism by which Bcl-2 family members may be regulated is ubiquitin/proteasome-mediated degradation. T cells have specialized proteasomes called "immunoproteasomes" that contain three interferon-g-inducible catalytic immunosubunits: LMP2, LMP7 and MECL-1. We have recently observed that T cells from immunoproteasome-deficient mice lacking both MECL-1 and LMP7 exhibit exaggerated apoptosis after in vivo activation by super-antigen. Conversely, over-expression of LMP7 in normal super-antigen activated T cells significantly suppresses apoptosis. Taken together, these results suggest that immunoproteasomes play a role in regulating the magnitude of apoptosis of activated T cells. Given the central role of Bcl-2 family members in mediating activated T cell apoptosis, and that several Bcl-2 family members are proteasome substrates, we hypothesize that immunoproteasomes regulate the magnitude of Bim-driven apoptosis of activated T cells through degradation of Bcl-2 family members. We will investigate this hypothesis in the following specific aims: 1. Determine the role of immunoproteasomes in the degradation and function of pro-apoptotic Bcl-2 family members. This aim will involve the generation of triple knockout mice (Bim-/-/MECL- 1-/-/LMP7-/-) to determine if Bim is required for the pro-apoptotic effect of immunoproteasome deficiency, and this aim will also assess the impact of immunoproteasome deficiency on the function and degradation of pro- apoptotic Bcl-2 family members in activated T cells. 2. Identify structural and functional features of immunoproteasomes that are critical for their anti-apoptotic activity. This aim will involve testing the ability of active site and assembly mutants of LMP7 and related proteasome subunits to modulate apoptosis of activated T cells, and determine whether immunoproteasome expression in normal activated T cells correlates with apoptotic activity. In the long-term, elucidation of molecular mechanisms underlying the role of immunoproteasomes in activated T cell apoptosis will further our understanding of the development of immune memory, and potentially identify targets for enhancing apoptosis of auto-reactive T cells involved in the pathogenesis of autoimmune disease, or suppressing apoptosis of T cells that could contribute to control of infectious or malignant disease. An immune response to a "threat" (such as infection or tumor) must be controlled in such a way as to provide memory to protect from similar threats in the future (immunity), while at the same time preventing unwanted damage to one's self (autoimmunity). The goal of this project is to improve our understanding of the molecules that participate in this control system, which could lead to better treatments of diseases in which this system is self-defeating or breaks down (infections, cancer, or autoimmune disease).