Mechanisms of ubiquitin pathway activation and function

 描述(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，此类疾病在全球范围内的患病率正在上升。根据疾病控制和预防中心的数据，目前有710万儿童患有哮喘(美国肺脏协会，2012年)9，其他许多儿童患有其他过敏性疾病。迫切需要针对TH2细胞因子产生T细胞的新疗法，同时保持免疫系统的其他手臂不变。这项建议的重点是Ndfip1，一种我们发现限制TH2细胞分化和过敏性炎症的蛋白质，以及E3泛素连接酶瘙痒。Ndfip1限制TH2的分化，部分是通过促进JunB的Itch泛素化。然而，很明显，Ndfip1的作用远远超出了这种相互作用。我们的新数据开始探索Ndfip1是如何工作的。这些数据揭示了泛素途径激活的新模式，这表明治疗策略可能对治疗哮喘等过敏性疾病有用。我们的初步数据为瘙痒和其他密切相关的家庭成员的激活定义了一个新的范例。在这项提案中，我们将回答一些重要的悬而未决的问题，解释Ndfip1如何激活瘙痒和相关的E3(目标1)。这些研究将确定单个WW结构域在解除瘙痒自身抑制、激活Ndfip1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，我们将鉴定和分析Ndfip1/Itch泛素化的底物。泛素途径的底物鉴定一直是个谜。然而，最近开发的工具现在使这成为可能(见目标2)。在这里，我们将使用两种新的可用富集法来确定底物，同时也测定泛素化赖氨酸残基(S)。我们已经测试了这两种技术，并已经确定了几种有前景的新底物，包括TCF1和PKCA。在目标3中，我们将测试这些底物和其他新发现的底物在体外和体内T细胞增殖、TH2分化和IL-4产生方面的作用。此外，我们将使用哮喘小鼠模型来确定治疗哮喘的可行性 以此为靶点治疗哮喘的治疗方法。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体如何被激活以及它们如何调节过敏性疾病的机制基础。这些信息将导致新的技术进步，同时为合理的治疗设计定义变构相互作用

 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