Structural basis of regulation of IRF

描述(由申请人提供)： 该项目的长期目标是了解干扰素调节因子(IRF)转录因子家族调控的结构基础。IRF-3是一个家族成员，具有抗病毒开关的功能。在未感染的细胞中，IRF-3以自身抑制的形式在细胞质中结构性表达。在病毒感染后，IRF-3被磷酸化和寡聚化，进入细胞核并通过与辅活化子CBP/p300的必要相互作用转录激活天然免疫程序。人类疱疹病毒81Kaposi肉瘤相关疱疹病毒(HHV-8/KSHV)通过表达一种病毒形式的IRF，vlRF-1，通过隔离CBP/p300来干扰IRF-3的激活，从而抵消宿主的防御。该提案调查了通过磷酸化和CBPIp300相互作用激活IRF-3的结构基础，以及通过病毒基因产物vlRF-I使IRF-3失活的结构基础。在初步研究中，确定了自抑制的IRF-3反式激活结构域的晶体结构。结构分析显示，Smad蛋白家族与Smad家族蛋白具有显著的结构同源性和潜在的机制相似性，Smad家族蛋白是转化生长因子β途径的转录中介。该结构为研究IRF-3的磷酸化激活机制提供了框架。此外，IRF-31CBP络合物的衍射性晶体是可用的，这将导致结构的确定。提出了三个具体目标。首先，我们将对IRF-3磷酸化的结构基础进行研究，以揭示IRF-3激活状态中的低聚状态、低聚界面和磷酸化的结构作用。其次，将研究IRF-3与CBP/p300相互作用的结构基础，以揭示转录调控中的特异性识别机制。最后，将研究病毒vlRF-1与宿主IRF-3竞争CBPIp300相互作用的结构基础，以揭示竞争机制。我们将综合运用X射线结晶学、分析超速离心法、等温滴定量热法、质谱仪、细胞生物学和其他生化技术来研究这些问题。这些研究将为深入了解IRF信号的分子机制，为合理设计对抗病毒致病的药物提供可能的靶点。

DESCRIPTION (provided by applicant): The long-term goal of the project is to understand the structural basis of regulation of the interferon regulatory factor (IRF) family of transcription factors. IRF-3 is a family member that functions as an anti-viral switch. In uninfected cells, IRF-3 is constitutively expressed in the cytoplasm in the autoinhibited form. Upon virus infection, IRF-3 becomes phosphorylated and oligomerized, which enters the nucleus and transcriptionally activates the innate immune program through an essential interaction with the coactivator CBP/p300. The human herpesvirus 81Kaposi Sarcoma-associated herpesvirus (HHV-8/KSHV) counteracts the host's defense by expressing a viral form of IRF, vlRF-1 ,-that.interferes IRF-3 activation through sequestering CBP/p300. The proposal investigates the structural basis of IRF-3 activation by phosphorylation and CBPIp300 interaction, as well as IRF-3 inactivation by the viral gene product vlRF-I. In the preliminary studies, crystal structure of the autoinhibited IRF-3 transactivation domain was determined. Structure analysis revealed a remarkable structural homology, and potential mechanistic similarity, with the Smad family of proteins, which are transcriptional mediators of the transforming growth factor beta pathway. The structure provides a framework for investigating the mechanism of IRF-3 phospho-activation. Furthermore, diffraction quality crystals of the iRF-31CBP complex are available, which will lead to a structural determination. Three specific aims are proposed. First, the structural basis of IRF-3 phosphoactivation will be investigated to reveal the oligomeric state, oligomeric interface and structural role of phosphorylation in the activate state IRF-3. Second, the structural basis of IRF-3 interaction with CBP/p300 will be investigated to reveal the mechanism of specific recognition in transcriptional regulation. Finally, the structural basis of viral vlRF-1 competing with host IRF-3 for CBPIp300 interaction will be investigated to reveal the mechanism of competition. A combinatorial approach using X-ray crystallography, analytical ultracentrifugation, isothermal titration calorimetry, mass spectrometry, cellular biology and other biochemical techniques will be employed to investigate these questions. These studies will provide insight into the molecular mechanism of IRF signaling, providing possible targets for rational drug design to combat viral pathogenesis.