FBXO6与糖基化Ero1L相互作用在内质网应激中的分子机制及其生物学意义

FBXO6 is the substrate recognition component of the ubiquitin ligase complex SCF (Skp1/Cullin1/F-box), which involved in cell cycle regulation, endoplasmic-reticulum-associated protein degradation (ERAD), and tumor occurrence and development. Recently, utilizing protein immunoprecipitation purification combined with LC−MS, we firstly identified 29 glycoproteins highly interacted with FBXO6 in a variety of cell lines. Furthermore, we found that FBXO6 interacted with glycosylated Ero1L, an endoplasmic reticulum stress core protein, and thus modulated Ero1L protein levels. This project will be based on these original findings, using FBXO6-Ero1L interaction as an entry point, utilizing Fbxo6 gene deficient mice cells and tissues, trying to clarify the molecular mechanisms of the regulation of FBXO6 on Ero1L protein, and thus to reveal the biological significance of FBXO6 in endoplasmic reticulum stress reactions. This study will help to further reveal the new biological functions of FBXO6 and a new mechanism of endoplasmic reticulum stress-induced apoptosis. This study will also provide a possible new molecular target for the therapy of diseases that utilizing endoplasmic reticulum stress as a common molecular mechanism.

FBXO6是泛素蛋白连接酶复合物SCF的底物识别组分，参与调控细胞周期以及糖蛋白的内质网相关降解(ERAD)，并与肿瘤的发生、发展有着密切的联系。我们最近采用蛋白质免疫沉淀结合液相质谱技术(IP-LC/MS)，首次在多种细胞株中鉴定了29个与FBXO6高度结合的糖蛋白，其中发现FBXO6与内质网应激核心糖基化蛋白Ero1L存在相互作用，进而调控Ero1L的蛋白水平。本项目将在这些原创性发现的基础上，以FBXO6和糖基化Ero1L蛋白的相互作用为切入点，以Fbxo6基因缺失小鼠细胞和组织为研究对象，试图阐明FBXO6对Ero1L蛋白的调控作用和分子机制，进而揭示FBXO6在内质网应激反应中的生物学意义。本研究将有益于深入揭示FBXO6新的生物学功能、内质网应激诱导细胞凋亡的新机制，并为以内质网应激为共通分子机制的常见疾病的治疗提供可能的新的分子靶标。

F-box蛋白是泛素蛋白连接酶复合物SCF的底物识别组分，通过募集并促进特异性底物蛋白的泛素化降解来发挥其生物学功能。我们前期利用免疫共沉淀与生物质谱联合运用(IP-MS)发现了Ero1L为FBXO6相互作用糖蛋白。通过gain-of-function和loss-of-function的方法，我们此次研究发现FBXO6能在蛋白水平调控Ero1L的半衰期，促进其泛素化降解。FBXO6介导的Ero1L的泛素化降解参与调控内质网应激导致的细胞凋亡过程。FBXO2与FBXO6都属于含有糖蛋白识别区域的F-box家族成员，我们采用蛋白质免疫沉淀结合液相质谱技术，首次系统性鉴定了528个FBXO2特异识别糖蛋白，阐明了FBXO2对糖基化胰岛素受体INSR的识别与降解的分子机制，发现肝脏特异性过表达FBXO2能促进C57BL/6小鼠高血糖症并导致了胰岛素抵抗，在db/db小鼠肝脏里特异性敲除FBXO2则能增强胰岛素敏感性。发现IKKβ/NF-κB炎症信号通路能直接调控FBXO2的表达。这些研究揭示了FBXO6调控内质网应激导致的细胞凋亡的分子机制，明确了FBXO2降解INSR对小鼠的生物学意义，为临床上治疗以内质网应激为发病基础的重大疾病，提供了重要的实验基础和分子靶标。