Immune regulation by SLAT, a Novel Th2-Expressed Protein

描述（由申请人提供）：我们最近分离了一种新的蛋白质，SLAT，一个新的Rac特异性激活剂亚家族的成员。SLAT在Th 2细胞中上调，抗原刺激诱导其免疫突触定位和ZAP- 70缔合，导致TCR募集和ZAP-70活化减少。我们的两个，非排他性的。工作假设是：1）SLAT通过其结合ZAP-70抑制激酶的TCR募集和/或活化，从而以促进Th 2分化、扩增和/或活化的方式调节TCR信号传导的质量;和2）SLAT和/或其替代产物SLAT 2在Th 2细胞中作为Cdc 42/Rac特异性GEF起作用;这种活性在决定Th 2细胞中IS和脂筏簇集的不同质量以及因此下游信号中起着非冗余和独特的作用。我们将解决这些假设，并通过探索四个目标来分析SLAT的表达，功能和调节的其他方面：1）使用原代小鼠T细胞、成像和生化技术、不同的刺激物和受体阻断抗体，我们将分析SLAT/SLAT 2 mRNA或蛋白的表达，研究它们的细胞内定位，绘制膜/IS定位所必需的残基，并评估这种本地化的意义。2)我们将对SLAT 1-小鼠进行详细分析，包括Th 1/Th 2分化、近端和下游信号传导事件、抗体和T细胞应答以及对与Th 1或Th 2表型占优势相关的实验性疾病的易感性。3)我们将探索SLAT和ZAP-70（或Syk）之间的关联的生物学相关性，通过绘制相互作用位点，并评估相互作用缺陷的SLAT或ZAP-70/Syk突变体，或显性负ZAP-70/Syk，对Th 1/Th 1的发展和激活的影响。4)我们将使用生物化学和遗传学方法，包括SLAT 1+小鼠或用SLAT突变体重建的T细胞，来绘制负责GEF活性的SLAT残基，研究调节SLAT的GEF活性的机制，并确定该活性对SLAT的Th 2-偏斜效应和对Th 2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.