Nedd4-family adaptors and their regulation of T cell function

Nedd4 家族接头及其对 T 细胞功能的调节

• 批准号: 9214304
• 负责人: Paula Maria Oliver
• 金额: $ 42万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-04 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9214304
• 关键词: Acute; Allergic Reaction; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Binding Proteins; Biological Assay; Biology; CRISPR/Cas technology; Cell Count; Cell Line; Cell physiology; Cells; Chronic; Collaborations; Complex; Cytokine Receptors; Cytokine Signaling; DNMT3a; Data; Dependence; Development; Disease; Endocytosis; Enzymes; FOXP3 gene; Family; Family member; Frequencies; Funding; Generations; Genetic Transcription; Grant; Half-Life; Hand; Homeostasis; Immune response; Immune system; Immunity; In Vitro; Infection; Inflammation; Interleukin-2; Interleukin-4; Knockout Mice; Mass Spectrum Analysis; Mediating; Membrane; Methods; Methylation; Methyltransferase; Mus; Pathway Analysis; Pathway interactions; Peripheral; Physiological; Process; Production; Proteins; Proteome; Pruritus; Receptor Signaling; Regulatory T-Lymphocyte; Research; Research Personnel; Role; Signal Transduction; T cell regulation; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Time; Transformed Cell Line; Ubiquitin; Validation; ZAP-70 Gene; autoinflammatory; base; bisulfite sequencing; cytokine; design; early onset; in vivo; novel; pathogen; premature; prevent; public health relevance; receptor; response; tool; transcription factor; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Regulatory T cells keep the immune system in check and limit collateral damage during pathogen invasion. These cells are required to prevent the development of autoimmune and autoinflammatory disorders, protect the host against allergic reactions, and control the immune response during acute and chronic infections. Now, over a decade since FoxP3 was revealed as the defining transcription factor of the majority of regulatory T cells (known as FoxP3+ Tregs), we are beginning to have a good understanding of the transcriptional profiles of these cells and their dependence on signals emanating from IL-2 and/or T cell receptors. In contrast, little is known about how these cells are regulated by ubiquitin cascades that promote signaling, endocytosis or degradation of the many proteins expressed by regulatory T cells. We hypothesize that Ndfip1 and Ndfip2 promote the activation of Itch, and related E3 ubiquitin ligases to regulate Treg homeostasis and function. Since this grant was initially funded in 2011, we have defined key biologic processes that are regulated by Ndfip1, a small membrane bound protein that activates Itch and related Nedd4-family E3 ubiquitin ligases. However, due to a lack of techniques that would allow us to define physiologically relevant substrates of these ubiquitin pathways, our studies were mostly descriptive. We have spent the past year trying to overcome these obstacles, designing strategies that could be used to screen for substrates. These efforts have been extremely successful and we are now poised to reveal key aspects defining how these ubiquitin complexes function. We are one of the first labs to define substrates that are ubiquitylated in T cells and the first to do so in primary cells rather than in cell lines. To accomplish this, we hav needed to 1) optimize the anti-K-e-GG enrichment strategy (as it requires a very high number of cells and to date has thus only been accomplished using transformed cell lines), 2) devise a new strategy using tandem ubiquitin binding entities (TUBEs) to enrich for ubiquitylated substrates, and 3) found ways to pair these approaches with additional approaches to increase our confidence as we select potential substrates for validation. Having these approaches in hand, we are now ready to expand our studies to explore other E3s and adaptors that are expressed in Tregs. Our aims are to 1) determine whether and how Ndfip1 limits TCR signaling and the frequency of eTregs, 2) determine whether and how Ndfip1 promotes Treg function and FoxP3 stability, and 3) to determine the relative contributions of Ndfip1 and Ndfip2 and their cognate E3s in Treg homeostasis and function. To accomplish these aims, I have assembled a team of investigators with expertise in Treg biology and ubiquitin pathway analysis (Oliver), mass spectrometry (Seeholzer), FoxP3 methylation (Wells), and the generation of mice using CRISPR/Cas9 (Henao-Majia). Together, we are poised to make significant contributions towards understanding how ubiquitin regulates key aspects of Treg biology.

 描述（由申请人提供）：调节性 T 细胞控制免疫系统并限制病原体入侵期间的附带损害。这些细胞需要预防自身免疫和自身炎症性疾病的发展，保护宿主免受过敏反应，并在急性和慢性感染期间控制免疫反应。现在，自从 FoxP3 被揭示为大多数调节性 T 细胞（称为 FoxP3+ Tregs）的决定性转录因子以来，我们开始对这些细胞的转录谱及其对 IL-2 和/或 T 细胞受体发出的信号的依赖性有了很好的了解。相比之下，人们对这些细胞如何受到泛素级联的调节知之甚少，而泛素级联促进调节性 T 细胞表达的许多蛋白质的信号传导、内吞作用或降解。我们假设 Ndfip1 和 Ndfip2 促进 Itch 和相关 E3 泛素连接酶的激活，从而调节 Treg 稳态和功能。自 2011 年首次资助以来，我们已经定义了由 Ndfip1 调节的关键生物过程，Ndfip1 是一种小膜结合蛋白，可激活 Itch 和相关的 Nedd4 家族 E3 泛素连接酶。然而，由于缺乏能够让我们定义这些泛素通路的生理相关底物的技术，我们的研究大多是描述性的。在过去的一年里，我们一直在努力克服这些障碍，设计可用于筛选底物的策略。这些努力非常成功，我们现在准备揭示定义这些泛素复合物如何发挥作用的关键方面。我们是最早定义 T 细胞中泛素化底物的实验室之一，也是第一个在原代细胞而不是细胞系中这样做的实验室。为了实现这一目标，我们需要 1) 优化抗 K-e-GG 富集策略（因为它需要非常大量的细胞，因此迄今为止只能使用转化的细胞系来完成），2) 使用串联泛素结合实体 (TUBE) 设计一种新策略来富集泛素化底物，3) 找到将这些方法与其他方法配对的方法，以增加我们在选择潜在底物时的信心。 验证。有了这些方法，我们现在准备扩大我们的研究，探索在 Tregs 中表达的其他 E3 和适配器。我们的目标是 1) 确定 Ndfip1 是否以及如何限制 TCR 信号传导和 eTreg 的频率，2) 确定 Ndfip1 是否以及如何促进 Treg 功能和 FoxP3 稳定性，以及 3) 确定 Ndfip1 和 Ndfip2 及其同源 E3 在 Treg 稳态和功能中的相对贡献。为了实现这些目标，我组建了一个研究团队，他们在 Treg 生物学和泛素通路分析 (Oliver)、质谱 (Seeholzer)、FoxP3 甲基化 (Wells) 以及使用 CRISPR/Cas9 产生小鼠 (Henao-Majia) 方面拥有专业知识。我们将共同为理解泛素如何调节 Treg 生物学的关键方面做出重大贡献。