ARF-6潜在效应因子UNC-16/JIP3在囊泡循环运输中的调控功能研究

ARF6介导的非笼形蛋白依赖内吞 (CIE) 膜蛋白的囊泡循环运输与血糖调控、免疫受体应答等生物学过程密切相关。当前对ARF6不同效应因子在循环调控中的功能缺乏深入了解，且体外培养细胞研究成果不能完全反映生理状态下ARF6功能机制。近年模式生物秀丽线虫在体囊泡循环运输研究已有较好理论和技术基础，我们的前期工作预示ARF-6潜在效应因子UNC-16/JIP3参与调控CIE膜蛋白hTAC循环，unc-16突变导致肠细胞中hTAC异常累积表型。本课题将研究UNC-16亚细胞定位机制及其膜蛋白调控特异性，阐明UNC-16与ARF-6、RAB-10、RAB-5间遗传学关系，并进一步探讨UNC-16 对ARF-6(GTP)、RAB-5(GTP) 活性态的调控机制。本项目期望能系统研究整体动物中UNC-16循环调控机理，加深对ARF6循环路径协同调控机制的了解，为循环运输异常相关疾病的治疗提供科学依据。

ARF6 mediated clathrin-independent endocytic recycling plays key roles in many biological processes including blood sugar levels control, immune response. Due to limited approaches and tools in the field, much is unknown with respect to the recycling regulatory functions of ARF6 effectors. Furthermore, most studies had been done with culture cells, which do not always reveal the mechanisms in vivo. Recently, recycling trafficking studies using model organism C. elegans have been systematically deployed and well appreciated, and our preliminary works found that CIE cargo hTAC-GFP accumulates in enlarged vesicles in the intestine of unc-16 mutants, linking UNC-16/JIP3 to clathrin-independent endocytic recycling process. To better understand UNC-16 regulatory mechanisms in recycling in vivo, we propose to analyze UNC-16 subcellular localization regulation and the nature of UNC-16’s regulatory role in cargo recycling. Using genetic epistasis analysis we will determine the relationship of UNC-16 to ARF-6, RAB-10, and RAB-5. Next, we will check if UNC-16 can influence GTP-Occupancy of ARF-6 or RAB-5. If we do find that ARF-6(GTP) levels and/or PIP2 levels are affected in unc-16 mutants we will determine if UNC-16 binds to CNT-1. If RAB-5(GTP) levels are elevated in unc-16 mutants, we will assay for effects of loss of UNC-16 on TBC-2 localization, and potential binding of UNC-16 to TBC-2. Our proposed studies are expected to explore the recycling regulation mechanisms of UNC-16 in whole animal, defining mechanistic details of how ARF6 involved endocytic recycling works. Our studies can be profoundly important in the design of strategies to combat multiple diseases including type II diabetes and cancer.