Understanding How the Ubiquitin Ligase CBL Regulates Innate Immune Responses

项目摘要/摘要 这是一份申请美国国立卫生研究院R01研究项目资助的申请书。我们的长期目标是确定如何 结核分枝杆菌(Mtb)扰乱宿主免疫反应，在人类中建立感染，并 利用这些信息开发更好的结核病疫苗和治疗方法。在我们之前的工作中，我们使用了 蛋白质组学，以系统地确定分泌的结核分枝杆菌蛋白与人类蛋白之间的相互作用，以及 发现了数百个高度特定的相互作用的网络，这些相互作用可能在结核分枝杆菌中发挥重要作用 致病机理和寄主防御。我们开始了对互动网络的初步遗传分析， 发现了一种新的毒力因子LpqN，以及其可能的宿主靶标泛素连接酶CBL。这个 这项应用的具体目标是研究CBL调节先天性免疫信号的机制。 在巨噬细胞和小鼠体内。 在目标1中，我们将使用生化分析来检验CBL泛素化IRF3和/或IRF7的假设， 以它们为目标进行降解，从而抑制抗病毒反应。如果这一假设是错误的，我们将 采用更全球化的方法来识别CBL基材。 在目标2中，我们将确定毒力因子LpqN干扰CBL功能的机制 促进MTB增长。在目标3中，我们将探索我们已确定的一种新的磷酸化事件的功能 在CBL的丝氨酸450上。这些发现将为未来的研究铺平道路，这些研究将试图揭示 这些寄主因子促进免疫的分子机制，并可能提示 这些因素可以被操控，以获得治疗益处。

PROJECT SUMMAY/ABSTRACT This is an application for an NIH R01 Research Project Grant. Our long-term goal is to determine how Mycobacterium tuberculosis (Mtb) disrupts the host immune response to establish infection in humans, and to use this information to develop better vaccines and therapies for tuberculosis (TB). In our prior work, we used proteomics to systematically define the interactions between secreted Mtb proteins and human proteins, and discovered a network of several hundred highly-specific interactions that potentially play important roles in Mtb pathogenesis and host defense. We began an initial genetic analysis of the interaction network, and discovered both a novel virulence factor LpqN, as well as its probable host target, the ubiquitin ligase CBL. The specific goal of this application is to study the mechanisms by which CBL regulates innate immune signaling in macrophages and in mice. In Aim 1 we will use biochemical analyses to test the hypothesis that CBL ubiquitylates IRF3 and/or IRF7, targeting them for degradation and thus suppressing antiviral responses. If this hypothesis is incorrect we will take more global approaches to identifying CBL substrates. In Aim 2 we will determine the mechanism by which the virulence factor LpqN disrupts CBL function to promote Mtb growth. In Aim 3 we will explore the function of a novel phosphorylation event we have identified on serine 450 of CBL. These findings will pave the way for future studies that will seek to uncover the molecular mechanisms by which these host factors contribute to immunity and may suggest ways that the factors can be manipulated for therapeutic benefit.