Immune regulation by SLAT, a Novel Th2-Expressed Protein

描述(由申请人提供)：我们最近分离出一种新的蛋白质SLAT，它是RAC特异性激活子的一个新的亚家族的成员。SLAT在Th2细胞中上调，抗原刺激诱导其免疫突触定位和ZAP-70结合，导致TCR募集和ZAP-70激活减少。我们两个，非排他性的。工作假说是：1)SLAT通过其结合的ZAP-70抑制激酶的TCR募集和/或激活，从而以促进Th2分化、扩张和/或激活的方式调节TCR信号的质量；以及2)SLAT和/或其替代产物SLAT2在Th2细胞中作为CDC42/RAC特异性的全球环境基金发挥作用；这一活性在决定IS和脂筏聚集的不同质量以及因此在Th2细胞中的下游信号方面发挥着非冗余和独特的作用。我们将针对这些假说，通过探索四个目标来分析SLAT的表达、功能和调控的其他方面：1)利用原代小鼠T细胞、成像和生化技术、不同的刺激和受体阻断抗体，我们将分析SLAT/SLAT2的mRNA或蛋白的表达，研究它们在细胞内的定位，定位膜/IS定位所必需的残基，并评估这种定位的意义。2)我们将对SLAT1“小鼠进行详细的分析，包括Th1/Th2分化、近端和下游信号事件、抗体和T细胞反应，以及与Th1或Th2表型占优势相关的实验性疾病的易感性。3)通过绘制SLAT与ZAP-70(或Syk)的相互作用位点图，并评估相互作用缺陷的SLAT或ZAP-70/Syk突变体或显性阴性ZAP-70/Syk对Th1/Th1的发育和激活的影响，我们将探索SLAT与ZAP-70(或Syk)之间的生物学相关性。4)我们将利用生物化学和遗传学方法，包括SLAT1小鼠或用slat突变体重组的T细胞，定位与SLAT活性有关的SLAT残基，研究调节SLAT活性的机制，并确定该活性对SLAT的Th2扭曲效应和Th2细胞中IS的不同组织的意义。这些研究将表征一种新的、多功能的TCR-近端蛋白。

DESCRIPTION (provided by applicant): We recently isolated a novel protein, SLAT, member of a novel subfamily of Rac-specific activators. SLAT is upregulated in Th2 cells and antigen stimulation induces its immune synapse localization and ZAP- 70 association, leading to reduced TCR recruitment and activation of ZAP-70. Our two, non-exclusive. working hypotheses are: 1) SLAT, via its association ZAP-70, inhibits the kinase's TCR recruitment and/or activation, thereby modulating the quality of TCR signaling in a manner that promotes Th2 differentiation, expansion and/or activation; and 2) SLAT and/or its alternative product, SLAT2, functions in Th2 cells as a Cdc42/Rac-specific GEF; this activity plays a non-redundant and unique role in determining the distinct quality of the IS and lipid raft clustering and, consequently, downstream signals, in Th2 cells. We will address these hypotheses and analyze other aspects of the expression, function and regulation of SLAT by exploring four aims: 1) Using primary mouse T cells, imaging and biochemical techniques, different stimuli, and receptor-blocking antibodies, we will analyze the expression of SLAT/SLAT2 mRNA or protein, study their intracellular localization, map residues essential for the membrane/IS localization, and assess the significance of this localization. 2) We will conduct a detailed analysis of SLAT1" mice, including Th1/Th2 differentiation, proximal and downstream signaling events, antibody and T cell responses, and susceptibility to experimental diseases that are associated with predominance of the Th1 or Th2 phenotype. 3) We will explore the biological relevance of the association between SLAT and ZAP-70 (or Syk) by mapping the interaction sites, and assessing the effects of interaction-deficient SLAT or ZAP-70/Syk mutants, or dominant negative ZAP-70/Syk, on Th1/Th1 development and activation. 4) We will use biochemical and genetic approaches, including SLAT1' mice or T cells reconstituted with SLAT mutants, to map the SLAT residues responsible for GEF activity, study mechanisms that regulate the GEF activity of SLAT, and determine the significance of this activity for the Th2-skewing effect of SLAT and for distinct organization of the IS in Th2 cells. These studies will characterize a novel, multifunctional TCR-proximal protein.