TRIM27 is a new negative regulator of CD4 T cells and Mast cells.

描述(申请人提供)：K通道KCa3.1是钙内流以及随后激活B、T和肥大细胞所必需的。我们的研究发现，T细胞受体(TCR)的激活导致KCa3.1的激活，途径是激活2类磷脂酰肌醇3激酶C2 Beta(PI3K-C2)，从而产生PI3P，这是随后激活哺乳动物组氨酸激酶-二磷酸核苷激酶B所必需的，然后激活KCa3.1。我们鉴定了含有蛋白27的三部分基序(TRIM27)是PI3K-C2和KCa3.1的一个新的负调控因子。TRIM27是一个大的蛋白质家族的成员，其特征是存在一个由环指、B盒和卷曲线圈(CC)结构域组成的三部分基序。在这项建议中，我们将确定TRIM27调节PI3K-C2的机制，其生理意义，以及随后在体外和体内作为KCa3.1负调节因子在淋巴细胞和肥大细胞激活中的作用。我们有证据表明，TRIM27发挥E3连接酶的功能，泛素化并抑制PI3K-C2。为了确定TRIM27调节PI3K-C2的机制(S)以及TCR激活如何影响这一机制，我们将在SA1中确定：(AI)TRIM27介导的PI3K-C2的泛素化类型；(Aii)TRIM27和PI3K-C2上泛素化的赖氨酸残基；以及(Aiii)TRIM27和PI3K-C2上介导它们结合的区域或结构域。在1B中，我们将确定：(Bi)TRIM27是否调节TCR刺激的PI3K-C2的S激酶活性和/或降解，或者TRIM27与PI3K-C2的结合是否受到TCR刺激的影响；(Biii)TRIM27是否被招募到免疫突触(IS)，它在免疫突触形成和/或TCR激活后募集PI3K-C2到IS中的作用；(Biv)TRIM27是否调节激活的T细胞中PI3P的水平，或者(BV)TCR刺激是否调节TRIM27的自素化或PI3K-C2的泛素化。在(C)中，将评估TRIM27刺激的PI3K-C2 SUMO化的作用。我们已经建立了TRIM27-/-小鼠，并有证据表明，在TRIM27-/-Th1细胞中，KCa3.1通道活性和TCR刺激的钙通量增加。在SA2(A)中，我们将把这些研究扩展到其他CD4辅助T细胞亚群，并确定从TRIM27-/-小鼠分离的细胞中Th2、Th17和Treg的分化和/或功能是否发生改变。在(B)中，我们将确定受TRIM27调控的下游信号通路，以及(C)TRIM27-/-小鼠是否易患自身免疫性疾病。在(D)中，我们将采取一种无偏见的方法来确定TRIM27泛素化和调控的其他靶点。我们发现，TRIM27具有负调控FceR1刺激的肥大细胞KCa3.1通道活性和钙离子通量的功能。我们将在SA3中确定：(A)PI3K-C2是否介导FceR1刺激的KCa3.1激活以及TRIM27是否调节这一过程；(B)TRIM27-/-肥大细胞的效应功能是否增加；(3C)TRIM27-/-肥大细胞中的信号通路是否被TRIM27调节；和/或(3D)TRIM27-/-小鼠是否对被动皮肤和全身过敏反应都有增强的过敏反应。

DESCRIPTION (provided by applicant): The K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B, T, and mast cells. Our studies have found that T cell receptor (TCR) activation leads to activation of KCa3.1 by activating the class 2 phosphatidylinositol 3 kinase C2 Beta (PI3K-C2¿) leading to the generation of PI3P, which is required for the subsequent activation of a mammalian histidine kinase, Nucleoside Diphosphate Kinase B, that then activates KCa3.1. We identified the Tripartate Motif containing protein 27 (TRIM27) as a new negative regulator of PI3K-C2¿ and KCa3.1. TRIM27 is a member of a large family of proteins characterized by the presence of a tripartite motif, consisting of a RING finger, B box, and coiled coil (CC) domains. In this proposal, we will determine the mechanisms whereby TRIM27 regulates PI3K-C2¿, its physiological significance, and subsequent role as a negative regulator of KCa3.1 in lymphocyte and mast cell activation in vitro and in vivo. We have evidence that TRIM27 functions an E3 ligase and ubiquitinates and inhibits PI3K-C2¿. To determine the mechanism(s) whereby TRIM27 regulates PI3K-C2¿ and how this is affected by TCR activation, we will determine in SA1, (Ai) the type of TRIM27 mediated ubiquitination of PI3K-C2¿; (Aii) the lysine residues on TRIM27 and PI3K-C2¿ that are ubiquitinated; and (Aiii) the regions or domains on TRIM27 and PI3K-C2¿ that mediate their association. In 1B, we will determine: (Bi) if TRIM27 regulates TCR stimulated PI3K-C2¿ 's kinase activity and/or degradation, or whether (Bii) association of TRIM27 with PI3K-C2¿ is affected by TCR stimulation; (Biii) whether TRIM27 is recruited to the immunological synapse (IS), its role in IS formation and/or the recruitment PI3K-C2¿ to IS following TCR activation; (Biv) whether TRIM27 modulates the PI3P levels in activated T cells, or (Bv) whether TRIM27 autoubiquitination, or ubiquitination of PI3K-C2¿ is modulated by TCR stimulation. In (C), the role of TRIM27 stimulated SUMOylation of PI3K-C2¿ will be assessed. We have generated TRIM27-/- mice and have evidence that KCa3.1 channel activity and TCR- stimulated Ca2+ flux are increased in TRIM27-/- Th1 cells. In SA2 (A), we will extend these studies to other CD4 helper T cell subsets, and determine whether Th2, Th17, and Treg differentiation and/or function are altered in cells isolated from TRIM27-/- mice. In (B), we will determine the downstream signaling pathways regulated by TRIM27, and (C) whether TRIM27-/- mice are predisposed to autoimmune disease. In (D) we will undertake a nonbiased approach to identify other targets of TRIM27 ubiquitination and regulation We found that TRIM27 functions to negatively regulate FceR1 stimulated KCa3.1 channel activity and Ca2+ flux in mast cells. We will determine in SA3: (A) if PI3K-C2¿ mediates FceR1 stimulated activation of KCa3.1 and whether this is modulated by TRIM27; (B) whether effectors functions of TRIM27-/- mast cells are increased; (3C) the signaling pathways regulated by TRIM27 in mast cells, and/or (3D) whether TRIM27-/- mice have an increased anaphylactic response to both passive cutaneous and systemic anaphylaxis.