酮戊二酸脱氢酶（OGDH）在TRIM15调控IFN-β信号通路中的功能及机制研究

Most members of TRIM (Tripartite motif) family of proteins are recognized as the products of interferon-stimulated genes (ISGs) and as immune signaling mediators. Various TRIM proteins mediate antiviral response and play a very important role in innate antiviral immunity. Studies showed that TRIM15 was a critical component of the RLR signaling and could positively regulate the production of interferon beta (IFN-β), but the molecular mechanism still keeps unclear. Our previous study had identified the interaction between TRIM15 and oxoglutarate dehydrogenase (OGDH). We also found that knockdown of OGDH enhanced Sendai virus-induced IFN-β production. However, further research is required to be done to elucidate the function and molecular mechanism of the interaction between OGDG and TRIM15 in regulating innate antiviral immunity. In this project, TRIM15 and OGDG will be studied by the methods of protein mass spectrometry technology, protein-protein interaction, proteins overexpression, siRNA knockdown, plaque forming unit test and Dual-luciferase assay. We hope to address the following questions: (1).The interaction between TRIM15 and OGDH, and the influence on the expression and post-translational modifications of OGDH by TRIM15; (2). The role of OGDH in the production of type I interferon (IFN-β) pathway; (3). The function and molecular mechanism to active the IFN-β signal pathways and its relevance to the interaction between TRIM15 and OGDH. This study will be useful to clarity the mechanism of TRIM15-mediated regulation of innate antiviral immunity, and contribute to the understanding of immune regulation and develop antiviral drug to some infectious disease in the future.

TRIM家族蛋白中许多成员是重要的干扰素刺激因子，在宿主的抗病毒天然免疫反应中扮演着重要作用。目前发现TRIM15蛋白可调控IFN-β的产生，但其具体分子细节并不清楚。我们通过串联亲和纯化技术发现TRIM15与宿主蛋白酮戊二酸脱氢酶（OGDH）存在相互作用，而且下调内源性OGDH表达后能增强仙台病毒诱导的IFN-β产生，但OGDH在TRIM15抗病毒天然免疫中功能及其分子机制需要进一步深入研究。本研究拟以TRIM15与OGDH蛋白相互作用为切入点，利用蛋白质质谱、蛋白互作、超表达、siRNA干扰、荧光素酶报告系统等方法和技术研究：（1）TRIM15与OGDH蛋白互作分子细节及其对OGDH表达与修饰的影响；（2）OGDH蛋白对IFN-β信号通路的调控作用；（3）TRIM15与OGDH互作在其调控IFN-β信号通路中功能与机制。该研究为进一步揭示TRIM15抗病毒天然免疫调控机制提供科学依据。

TRIM家族蛋白中许多成员是重要的干扰素刺激因子，在宿主的抗病毒天然免疫反应中扮演着重要作用。目前发现TRIM15蛋白可以调控IFN-β的产生，但其具体的分子细节并不清楚。本项目中，我们课题组通过在细胞水平过表达TRIM15能上调IFN-的产生，并能有效抑制模式病毒VSV-GFPd的增殖；通过免疫沉淀联合质谱技术钓取TRIM15的互作蛋白OGDH，疫沉淀实验免疫荧光定位观察进一步研究二者之间存在相互作用；阐明了TRIM15与OGDH相互作用的分子细节：TRIM15的PRY-SPRY结构域和C-C结构域是其与OGDH发生相互作用的关键结构域，而仅全长OGDH可以与TRIM15发生相互作用。进而通过沉默和过表达OGDH证实了OGDH具有负调控IFN-β产生的能力，其分子机制是：OGDH与RLR信号通路中关键接头蛋白STING(干扰素刺激因子, Stimulator of interferon gene(STING)相互作用，通过溶酶体途径降解内源性STING进而负调控IFN-β的产生，而且OGDH的aa601-1023是调控STING降解的关键区域；尽管沉默OGDH能增强MAVS与STING的相互作用，但并不是通过与MAVS竞争性与STING结合来影响下游干扰素的产生。OGDH在抗病毒天然免疫中的作用有待深入研究。同时我们研究发现过表达TRIM15可依赖溶酶体途径降解内源性OGDH。可见，TIRM15正向调控型I型干扰素产生可能通过其降解OGDH，进而抑制的OGDH负调控IFN-β产生相关。本研究证实了OGDH是一个负调控I型干扰素的蛋白，而其在TRIM15表现的正调控干扰素产生中的详细机制有待深入研究。