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

描述（由申请人提供）：K+通道KCa 3.1是Ca 2+内流和随后激活B、T和肥大细胞所必需的。我们的研究发现，T细胞受体（TCR）激活通过激活2类磷脂酰肌醇3激酶C2 β（PI 3 K-C2）导致产生PI 3 P而导致KCa 3.1激活，PI 3 P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KCa 3.1。我们鉴定了包含蛋白27（TRIM 27）的三部分基序作为PI 3 K-C2 <$和KCa 3.1的新负调节剂。TRIM 27是蛋白质大家族的成员，其特征在于存在由RING指、B盒和卷曲螺旋（CC）结构域组成的三联基序。在这项提案中，我们将确定TRIM 27调节PI 3 K-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负调节剂的作用。 我们有证据表明，TRIM 27功能E3连接酶和泛素化，抑制PI 3 K-C2？。为了确定TRIM 27调节PI 3 K-C2 <$$>的机制以及TCR活化如何影响这一机制，我们将确定SA 1中的（Ai）TRIM 27介导的PI 3 K-C2 <$$>泛素化的类型;（Aii）TRIM 27和PI 3 K-C2 <$的赖氨酸残基被泛素化;以及（Aiii）TRIM 27和PI 3 K-C2 <$介导其关联的区域或结构域。在1B中，我们将确定：（Bi）TRIM 27是否调节TCR刺激的PI 3 K-C2？的激酶活性和/或降解，或者（Bii）TRIM 27与PI 3 K-C2？的结合是否受到TCR刺激的影响;（Biii）TRIM 27是否被募集到免疫突触（IS），其在IS形成中的作用和/或TCR激活后将PI 3 K-C2？募集到IS;（Biv）TRIM 27是否调节活化T细胞中的PI 3 β水平，或（Bv）TRIM 27自身泛素化或PI 3 K-C2 β的泛素化是否受TCR刺激调节。在（C）中，将评估TRIM 27刺激的PI 3 K-C2？SUMO化的作用。 我们已经产生了TRIM 27-/-小鼠，并且有证据表明TRIM 27-/-Th 1细胞中KCa3.1通道活性和TCR刺激的Ca 2+通量增加。在SA 2（A）中，我们将这些研究扩展到其他CD 4辅助性T细胞亚群，并确定从TRIM 27-/-小鼠分离的细胞中Th 2、Th 17和Treg分化和/或功能是否发生改变。在（B）中，我们将确定由TRIM 27调节的下游信号传导途径，和（C）TRIM 27-/-小鼠是否易患自身免疫性疾病。在（D）中，我们将采用无偏倚的方法来鉴定TRIM 27泛素化和调节的其他靶标。我们发现TRIM 27具有负调节肥大细胞中FceR 1刺激的KCa 3.1通道活性和Ca 2+通量的功能。我们将在SA 3中确定：（A）如果PI 3 K-C2？介导FceR 1刺激的KCa 3.1的活化以及这是否被TRIM 27调节;（B）TRIM 27-/-肥大细胞的效应子功能是否增加;（3C）肥大细胞中由TRIM 27调节的信号传导途径，和/或（3D）TRIM 27-/-小鼠是否对被动皮肤和全身过敏反应具有增加的过敏反应。

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.