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

TRIM27 是 CD4 T 细胞和肥大细胞的新型负调节因子。

• 批准号: 8218480
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 32.96万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218480
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affect; Anaphylaxis; Applications Grants; Asthma; Autoimmune Diseases; B-Cell Activation; Binding; Boxing; CD4 Positive T Lymphocytes; Calcium-Activated Potassium Channel; Cells; Coiled-Coil Domain; Cutaneous; Development; Failure; Feedback; Fingers; Generations; Goals; Graft Rejection; Helper-Inducer T-Lymphocyte; Histidine; Human; Hypersensitivity; Immune system; In Vitro; Inflammation; Lead; Lymphocyte; Lysine; Mediating; Mus; Names; Nucleoside diphosphate kinase B; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Potassium Channel; Protein Family; Proteins; Receptor Activation; Recruitment Activity; Regulation; Regulatory T-Lymphocyte; Role; Signal Pathway; Signaling Molecule; Structure of thyroid parafollicular cell; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; TRIM Motif; Th1 Cells; Ubiquitination; Zinc; allergic response; follow-up; immunological synapse; immunological synapse formation; in vivo; inhibitor/antagonist; insight; mast cell; member; myotubularin; phosphatidylinositol 3-phosphate; phosphohistidine; prevent; protein-histidine kinase; response; ubiquitin-protein ligase

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. PUBLIC HEALTH RELEVANCE: Negative regulators of the adaptive and innate immune system, as well as mast cells, are critical to limit inflammation and to prevent allergic responses and autoimmune disease. Failure to down regulate these responses appropriately can lead to autoimmune disease and allergy, underscoring the critical role in understanding the signaling molecules that negatively regulate these cells. We have identified TRIM27 as a new negative regulator of CD4 T and mast cells. Understanding TRIM27's role in turning off these cells is likely to shed important insights into underlying mechanisms related to autoimmune disease and allergy.

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。