Mechanisms of ubiquitin pathway activation and function

泛素通路激活和功能的机制

• 批准号: 9254435
• 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: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9254435
• 关键词: Affect; Allergens; Allergic; Allergic Disease; Allergic inflammation; American Lung Association; Asthma; Binding; Biochemical; Biological Assay; Biology; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Centers for Disease Control and Prevention (U.S.); Charge; Child; Chronic; Chronic Disease; Complement; Complex; Data; Disease; Family; Family member; Fluorescence Resonance Energy Transfer; Goals; Human; Immune system; Individual; Inflammation; Inflammatory; Interleukin-4; Licensing; Lung; Lung Inflammation; Lysine; Mediating; Methods; Modeling; Mus; Mutation; PRKCA gene; Pathogenicity; Pathway interactions; Patients; Phenotype; Prevalence; Production; Proteins; Proteomics; Pruritus; Regulation; Research; Role; T-Cell Proliferation; T-Lymphocyte; Techniques; Testing; Th2 Cells; Therapeutic; Ubiquitin; Work; airway hyperresponsiveness; analytical method; arm; base; cytokine; design; disorder prevention; in vivo; innovation; insight; mouse model; novel; novel therapeutics; prevent; public health relevance; therapeutic target; tool; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Allergic diseases such as asthma are some of the most common chronic illnesses affecting children in the U.S. and such diseases are increasing in prevalence worldwide. Based on data from the Centers for Disease Control and Prevention, 7.1 million children are currently affected by asthma (American Lung Association 2012)9 and many others suffer from other atopic allergic diseases. New therapies are urgently needed that target TH2 cytokine producing T cells while leaving other arms of the immune system intact. This proposal focuses on Ndfip1, a protein that we discovered limits TH2 cell differentiation and allergic inflammation, and the E3 ubiquitin ligase Itch. Ndfip1 limits TH2 differentiation, in part by promoting Itch ubiquitylation of JunB. However, it is clear that Ndfip1 functions well beyond this interaction. Our new data begins to explore how Ndfip1 works. These data reveal novel modes of ubiquitin pathway activation that suggest therapeutic strategies that could be useful in treating allergic diseases such as asthma. Our preliminary data defines a new paradigm for the activation of Itch and other closely related family members. In this proposal, we will answer important unresolved questions explaining how Ndfip1 activates Itch and related E3s (Aim 1). These studies will define the function of individual WW domains in relief of Itch autoinhibition, Ndfip1 activation, and interaction with substrates, thus making allosteric therapeutic design possible. In Aim 2 we will identify and analyze substrates of Ndfip1/Itch ubiquitylation. Identifyig substrates of ubiquitin pathways has been enigmatic. However, tools have recently been developed that now make this possible (see Aim 2). Here we will use two newly available enrichment strategies to identify substrates while also determining the ubiquitylated lysine residue(s). We have tested both techniques and have already identified several promising new substrates including TCF1 and PKCa. In Aim 3, we will test these and other newly identified substrates for their roles in T cell proliferation, TH2 differentiation and IL-4 production assays n vitro and in vivo. Additionally, we will use mouse models of asthma to determine the feasibility of therapeutic targeting of this pathway to treat asthma. Thus, these studies will have a significant and long-term impact as they define the mechanistic basis of how ubiquitin complexes are activated and how they regulate allergic disease. Such information will result in new technological advances while defining allosteric interactions for rational therapeutic design

 描述（由申请人提供）：哮喘等过敏性疾病是影响美国儿童的一些最常见的慢性疾病，并且此类疾病在全球范围内的患病率正在增加。根据美国疾病控制与预防中心的数据，目前有 710 万儿童患有哮喘（美国肺脏协会 2012 年）9，还有许多其他儿童患有其他特应性过敏性疾病。迫切需要针对产生 TH2 细胞因子的 T 细胞的新疗法，同时保持免疫系统其他部分完好无损。该提案重点关注 Ndfip1（一种我们发现限制 TH2 细胞分化和过敏性炎症的蛋白质）和 E3 泛素连接酶 Itch。 Ndfip1 部分通过促进 JunB 的 Itch 泛素化来限制 TH2 分化。然而，很明显，Ndfip1 的功能远远超出了这种相互作用。我们的新数据开始探索 Ndfip1 的工作原理。这些数据揭示了泛素通路激活的新模式，提出了可用于治疗哮喘等过敏性疾病的治疗策略。我们的初步数据定义了激活 Itch 和其他密切相关的家庭成员的新范例。在本提案中，我们将回答重要的未解决问题，解释 Ndfip1 如何激活 Itch 和相关 E3（目标 1）。这些研究将定义各个 WW 结构域在缓解痒自动抑制、Ndfip1 激活以及与底物相互作用方面的功能，从而使变构治疗设计成为可能。在目标 2 中，我们将鉴定并分析 Ndfip1/Itch 泛素化的底物。识别泛素通路的底物一直是个谜。然而，最近开发的工具使这成为可能（参见目标 2）。在这里，我们将使用两种新可用的富集策略来识别底物，同时确定泛素化的赖氨酸残基。我们已经测试了这两种技术，并已经确定了几种有前途的新底物，包括 TCF1 和 PKCa。在目标 3 中，我们将在体外和体内测试这些和其他新鉴定的底物在 T 细胞增殖、TH2 分化和 IL-4 产生测定中的作用。此外，我们将使用哮喘小鼠模型来确定 该途径的治疗靶向治疗哮喘。因此，这些研究将产生重大和长期的影响，因为它们定义了泛素复合物如何激活以及它们如何调节过敏性疾病的机制基础。这些信息将带来新的技术进步，同时定义合理治疗设计的变构相互作用