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

描述（由申请人提供）：K+通道KCa3.1是Ca2+内流和随后的B、T和肥大细胞活化所必需的。我们的研究发现，T细胞受体（TCR）的激活通过激活2类磷脂酰肌醇3激酶C2 β （PI3K-C2¿）导致KCa3.1的激活，从而导致PI3P的产生，PI3P是随后激活哺乳动物组氨酸激酶核苷二磷酸激酶B所必需的，然后激活KCa3.1。我们发现Tripartate Motif containing protein 27 （TRIM27）是PI3K-C2¿和KCa3.1的一个新的负调控因子。TRIM27是一个大家族蛋白的成员，其特征是存在一个三方motif，由RING finger， B box和coil coil （CC）结构域组成。在这项提议中，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淋巴细胞和肥大细胞活化中作为KCa3.1负调节因子的作用。我们有证据表明TRIM27具有E3连接酶的功能，泛素化并抑制PI3K-C2¿。为了确定TRIM27调节PI3K-C2¿的机制以及TCR激活如何影响PI3K-C2¿，我们将在SA1中确定TRIM27介导的PI3K-C2¿泛素化的类型；（ii） TRIM27和PI3K-C2¿上泛素化的赖氨酸残基；以及（ii） TRIM27和PI3K-C2¿上介导其关联的区域或结构域。在1B中，我们将确定：（Bi） TRIM27是否调节TCR刺激的PI3K-C2¿的激酶活性和/或降解，或者（Bii） TRIM27与PI3K-C2¿的关联是否受到TCR刺激的影响；（Biii） TRIM27是否被募集到免疫突触（is），其在is形成中的作用和/或在TCR激活后将PI3K-C2¿募集到is；（Biv） TRIM27是否调节活化T细胞中的PI3P水平，或（Bv） TRIM27的自泛素化或PI3K-C2¿的泛素化是否被TCR刺激调节。在(C)中，将评估TRIM27刺激PI3K-C2¿的SUMOylation的作用。我们产生了TRIM27-/-小鼠，并有证据表明，KCa3.1通道活性和TCR刺激的Ca2+通量在TRIM27-/- Th1细胞中增加。在SA2 (A)中，我们将把这些研究扩展到其他CD4辅助性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TRIM27调节的下游信号通路，以及(C) TRIM27-/-小鼠是否易患自身免疫性疾病。在(D)中，我们将采用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的功能可以负调控肥大细胞中FceR1刺激的KCa3.1通道活性和Ca2+通量。我们将在SA3中确定：(A) PI3K-C2¿是否介导FceR1刺激的KCa3.1激活，以及这是否由TRIM27调节；(B) TRIM27-/-肥大细胞效应物功能是否增加；（3C）肥大细胞中TRIM27调节的信号通路，和/或（3D） TRIM27-/-小鼠是否对被动皮肤和全身过敏反应有增加的过敏反应。

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.