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: 8541082
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 4.34万
• 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/8541082
• 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.

描述（由申请人提供）：K+ 通道 KCa3.1 是 Ca2+ 流入以及随后 B、T 和肥大细胞激活所必需的。我们的研究发现，T 细胞受体 (TCR) 激活会导致 KCa3.1 激活。通过激活 2 类磷脂酰肌醇 3 激酶 C2 Beta (PI3K-C2¿) 导致 PI3P 的生成，这是后续激活所必需的哺乳动物组氨酸激酶，核苷二磷酸激酶 B，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 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，我们将确定其机制。因此 TRIM27 调节 PI3K-C2¿ 、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 具有 E3 连接酶功能，并且泛素化和抑制 PI3K-C2¿确定 TRIM27 调节 PI3K-C2 的机制以及 TCR 激活如何影响这一点，我们将在 SA1（Ai）中确定 TRIM27 介导的 PI3K-C2 泛素化的类型¿ (Aii) TRIM27 和 PI3K-C2 上的赖氨酸残基¿是泛素化的；和 (Aiii) TRIM27 和 PI3K-C2 上的区域或结构域在 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 TRIM27 是否与 PI3K-C2 关联 (Bii)受 TCR 刺激的影响；(Biii) TRIM27 是否被募集至免疫突触 (IS)，其在 IS 形成和/或募集 PI3K-C2 中的作用？ TCR 激活后的 IS；(Biv) TRIM27 是否调节活化 T 细胞中的 PI3P 水平，或 (Bv) TRIM27 是否自动泛素化或 PI3K-C2 泛素化？在 (C) 中，TRIM27 的作用刺激 PI3K-C2 的 SUMOylation。我们已经生成了 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，我们将把这些研究扩展到其他细胞。 CD4 辅助 T 细胞亚群，并确定从 TRIM27-/- 小鼠中分离的细胞中 Th2、Th17 和 Treg 分化和/或功能是否发生改变。在 (B) 中，我们将确定下游信号传导。 TRIM27 调节的途径，以及 (C) TRIM27-/- 小鼠是否易患自身免疫性疾病。 在 (D) 中，我们将采用无偏见的方法来确定 TRIM27 泛素化和调节的其他靶标。我们发现 TRIM27 具有负调节 FceR1 刺激的功能。肥大细胞中的 KCa3.1 通道活性和 Ca2+ 通量我们将在 SA3 中确定：(A) PI3K-C2¿介导 FceR1 刺激的 KCa3.1 激活，以及这是否受 TRIM27 调节；(B) TRIM27-/- 肥大细胞的效应器功能是否增加；(3C) 肥大细胞中 TRIM27 调节的信号传导途径，和/或 (3D) ）TRIM27-/- 小鼠对被动皮肤和全身过敏反应是否有增加的过敏反应。