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）家族转录因子调节的结构基础。IRF-3是作为抗病毒开关的家族成员。在未感染的细胞中，IRF-3以自抑制形式在细胞质中组成性表达。 在病毒感染后，IRF-3变得磷酸化和寡聚化，其进入细胞核并通过与辅激活因子CBP/p300的必要相互作用转录激活先天免疫程序。 人疱疹病毒81卡波西肉瘤相关疱疹病毒（HHV-8/KSHV）通过表达病毒形式的IRF（vlRF-1）来抵消宿主的防御，vlRF-1通过隔离CBP/p300来干扰IRF-3的激活。该提案研究了通过磷酸化和CBPIp 300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灭活IRF-3。 在初步研究中，确定了自抑制IRF-3反式激活结构域的晶体结构。 结构分析揭示了一个显着的结构同源性，和潜在的机制相似性，与Smad家族的蛋白质，这是转化生长因子β途径的转录介质。 该结构为研究IRF-3磷酸化活化机制提供了框架。 此外，可获得iRF-31 CBP复合物的衍射质量晶体，这将导致结构测定。提出了三个具体目标。首先，研究IRF-3磷酸化的结构基础，揭示IRF-3的寡聚状态、寡聚界面和磷酸化在激活状态IRF-3中的结构作用。其次，研究IRF-3与CBP/p300相互作用的结构基础，揭示IRF-3在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CBPIp 300相互作用的结构基础，以揭示竞争机制。将采用X射线晶体学、分析超离心、等温滴定量热法、质谱、细胞生物学和其他生物化学技术的组合方法来研究这些问题。这些研究将深入了解IRF信号传导的分子机制，为合理的药物设计提供可能的靶点，以对抗病毒的发病机制。

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.