Derlin-1在上皮细胞钠通道(ENaC)泛素化降解中的调控作用

The regulated activity of the distal nephron epithelial sodium channel (ENaC) is an important determinant of sodium balance, extracellular fluid volume, and blood pressure. The physiological significance of ENaC is illustrated most clearly by human genetic diseases in which channel mutations produce clinical defects in renal salt and water transport.The regulation of ENaC function really dependent on the balance of its Channel synthesis and channel degradation. However, our understanding of ENaC degradation in is quite incomplete. Recent studies show that derlin and its associated proteins play a critical role in ubiquitin mediated protein degradation. Our preliminary data suggest that the derlin complex effectively degrades ENaC in mouse cortical collecting duct (mCCD) cells. Our hypothesis is that the derlin complex is a key regulator of ubiquitin mediated ENaC degradation. The goal for this proposal is to identify components of the derlin complex that recognize and extract ENaC from the ER, and to identify the basis for their selective degradation of ENaC. The studies (in vivo) will be performed to examine whether derlin-1 complex participate in the regulation of ubiquitin-mediated epithelial sodium channel (ENaC) degradation using derlin-1 transgenic/knockout mice. We will test other four specific aims in mCCD cells (in vitro): (1) to define the role of derlin complex in ENaC degradation; (2) to examine the role of lysine-linkage of polyubiquitin chains in ENaC degradation; (3) to identify the processes mediating ENaC retro-translocation from the ER to the cytosol; and (4) to evaluate the ubiquitin E3 ligases that evoke ubiquitin-mediated ENaC degradation. Successful completion of this proposal will advance our understanding of molecular mechanism of ENaC regulation and provide a roadmap for identifying novel potential therapeutic targets to salt-sensitive hypertension.

肾脏远端肾单位上皮细胞钠离子通道 (ENaC) 的主要功能是介导钠离子的跨膜转运，调节水钠平衡并稳定血压。ENaC功能失调会引起人体血压调节障碍。ENaC表达转运的调控有赖于ENaC蛋白合成和蛋白降解的动态平衡，但是目前人们对ENaC降解的分子机制还知之甚少。Derlin-1及其相关蛋白在泛素化蛋白酶体降解途径中起着至关重要的作用。本课题的研究目的在于探讨derlin-1在ENaC泛素化蛋白酶体降解中的调控机制。我们将利用肾脏集合管上皮细胞derlin-1转基因及基因敲除小鼠模型，在体观察derlin-1对ENaC降解的调控作用；也将利用小鼠肾脏集合管上皮细胞进行体外实验，从四个方面 (详见研究内容) 来深入阐明derlin-1在介导ENaC泛素化降解过程中的分子机制。本课题的完成将为研究机体对钠离子重吸收开拓新的思路，为发现高血压新的治疗靶点奠定新的理论基础。

近几年，高血压是危害我国国民健康的主要疾病之一。据研究发现，至2013年，我国高血压患者已经突破2亿，而且每年还以1000万的速度增长。原发性高血压是遗传与环境因素相互作用所致的慢性疾病。高盐、激素紊乱等环境因素在很多情况下是通过机体不同细胞的膜蛋白来发挥作用。肾脏上皮钠离子通道 (epithelial sodium channel，ENaC) 正是重要的细胞膜蛋白之一。越来越多的研究表明，临床上发现的难治性高血压，包括盐敏感性高血压，也与ENaC的表达及功能的过度激活有关。ENaC主要表达在结肠，汗腺，涎腺导管和呼吸道的上皮细胞。在肾脏中，ENaC主要分布于远端肾单位的远曲小管和集合管，通过介导钠离子的跨膜转运对钠的重吸收起限速作用。Derlin1作为内质网降解相关蛋白，也是E3泛素连接酶的调节物，主要表达在细胞质中。本课题主要探究Derlin1参与ENaC泛素蛋白酶体途径降解的分子机制。在本课题中，我们首先发现了Derlin1能够在蛋白质水平降低ENaC的表达，但不影响ENaC mRNA的表达，提示我们Derlin1对ENaC是在翻译后水平进行调控的。通过免疫共沉淀实验(CO-IP) 我们发现Derlin1与ENaC具有直接的相互作用，而且二者结合在Derlin1的跨膜区和loop环。进一步研究表明Derlin1能够促进ENaC泛素化降解。K11位点为Derlin1介导ENaC泛素化降解的关键位点。同时质谱分析的结果提示E3酶HUWE1是Derlin1介导ENaC降解的关键酶。本课题深入了解ENaC降解的分子机制，为临床上寻找原发或继发性盐敏感性高血压的预防和治疗靶点具有重要的理论和指导意义。