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）：Ca2+ 流入以及随后 B、T 和肥大细胞的激活需要 K+ 通道 KCa3.1。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 Beta (PI3K-C2¿) 导致 KCa3.1 的激活，从而产生 PI3P，这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。我们鉴定出含有蛋白 27 (TRIM27) 的三部分基序作为 PI3K-C2¿ 和 KCa3.1 的新负调节因子。 TRIM27 是一个蛋白质大家族的成员，其特征是存在三部分基序，由环指、B 盒和卷曲螺旋 (CC) 结构域组成。在本提案中，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激活中作为 KCa3.1 负调节因子的作用。 我们有证据表明 TRIM27 发挥 E3 连接酶的作用，泛素化并抑制 PI3K-C2¿。为了确定 TRIM27 调节 PI3K-C2¿ 的机制以及 TCR 激活如何影响这一机制，我们将确定 SA1，(Ai) TRIM27 介导的 PI3K-C2¿ 泛素化的类型； (Aii) TRIM27 和 PI3K-C2¿ 上泛素化的赖氨酸残基； (Aiii) TRIM27 和 PI3K-C2 上介导它们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是否被募集至免疫突触(IS)、其在IS形成中的作用和/或TCR激活后将PI3K-C2募集至IS； (Biv) TRIM27 是否调节活化 T 细胞中的 PI3P 水平，或 (Bv) TRIM27 自身泛素化或 PI3K-C2 泛素化是否受 TCR 刺激调节。在 (C) 中，将评估 TRIM27 刺激 PI3K-C2 SUMO 化的作用。 我们已经培育出 TRIM27-/- 小鼠，并有证据表明 TRIM27-/- Th1 细胞中 KCa3.1 通道活性和 TCR 刺激的 Ca2+ 通量增加。在 SA2 (A) 中，我们将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 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，我们将采用无偏倚方法来确定 TRIM27 泛素化和调节的其他靶标。我们发现 TRIM27 具有负调节肥大细胞中 FceR1 刺激的 KCa3.1 通道活性和 Ca2+ 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 激活以及这是否受 TRIM27 调节； (B)TRIM27-/-肥大细胞的效应功能是否增强； (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。 公共卫生相关性：适应性和先天免疫系统以及肥大细胞的负调节因子对于限制炎症和预防过敏反应和自身免疫性疾病至关重要。如果不能适当下调这些反应，可能会导致自身免疫性疾病和过敏，这强调了了解负面调节这些细胞的信号分子的关键作用。我们已经确定 TRIM27 是 CD4 T 和肥大细胞的新负调节因子。了解 TRIM27 在关闭这些细胞中的作用可能有助于深入了解与自身免疫性疾病和过敏相关的潜在机制。