Role of Immunoproteasomes in Activated T Cell Apoptosis

描述(由申请人提供)：T细胞对同种抗原的克隆性扩增最终导致大部分扩增的T细胞通过程序性细胞死亡(细胞凋亡)而被删除，只有少数存活下来成为记忆细胞。这一过程对于在急性免疫反应终止后恢复T细胞动态平衡至关重要，而急性免疫反应可促进保护性免疫并阻止自身免疫。虽然现在有强有力的证据表明，Bcl-2家族的蛋白，特别是抗凋亡的Bcl-2和促凋亡的Bim在体内激活的T细胞的凋亡中起着重要的作用，但这些蛋白本身是如何被控制的还知之甚少。调控Bcl2家族成员的一种机制是泛素/蛋白酶体介导的降解。T细胞有特殊的蛋白酶体，称为“免疫蛋白酶体”，其中包含三种干扰素-g诱导的催化免疫亚群：LMP2、LMP7和MECL-1。我们最近观察到，缺乏MECL-1和LMP7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，在正常的超抗原激活的T细胞中过表达LMP7会显著抑制细胞凋亡。综上所述，这些结果提示免疫蛋白酶体在调节活化的T细胞的凋亡程度中起作用。鉴于Bcl2家族成员在介导活化的T细胞凋亡中的核心作用，以及几个Bcl2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Bim调节活化的T细胞的凋亡幅度。我们将从以下几个方面对这一假说进行研究：1.确定免疫蛋白酶体在促凋亡的Bcl-2家族成员的降解和功能中的作用。这一目标将包括建立三重基因敲除小鼠(Bim-/-/MECL-1-/LMP7-/-)，以确定免疫蛋白酶体缺陷是否需要Bim来发挥促凋亡作用，这一目标还将评估免疫蛋白酶体缺陷对活化T细胞中促凋亡的Bcl-2家族成员的功能和降解的影响。2.确定免疫蛋白酶体的结构和功能特征，这些特征对其抗凋亡活性至关重要。这一目标将包括检测LMP7和相关蛋白酶体亚单位的活性部位和组装突变体调节激活的T细胞凋亡的能力，并确定正常激活的T细胞中免疫蛋白酶体的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T细胞凋亡中作用的分子机制将加深我们对免疫记忆发展的理解，并有可能确定促进自身免疫性疾病发病机制中自身反应性T细胞凋亡的靶点，或抑制T细胞的凋亡，从而有助于控制感染性或恶性疾病。对“威胁”(如感染或肿瘤)的免疫反应必须得到控制，以便提供记忆，以保护未来免受类似威胁(免疫)，同时防止对自身的不必要损害(自身免疫)。这个项目的目标是提高我们对参与这一控制系统的分子的理解，这可能导致对这种系统自我挫败或崩溃的疾病(感染、癌症或自身免疫性疾病)的更好治疗。

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).