Mechanistic studies of prokaryotic and eukaryotic nitrate/nitrite transport

项目摘要 最近的研究表明，硝酸盐和亚硝酸盐分子对人类健康有深远的有益影响， 尽管自20世纪70年代以来它们被认为是癌症。为了最大限度地发挥其制药潜力，我们需要 首先了解硝酸盐和亚硝酸盐如何在人体内循环。这种循环取决于两个关键事件： 唾液腺中唾液酸转运蛋白介导的硝酸盐的主动积累，以及硝酸盐的摄取， 口腔中的细菌分泌亚硝酸盐。因此，硝酸盐/亚硝酸盐分子必须穿过不同的 在被人类使用之前，细胞膜多次。这些转运过程是由 一组叫做硝酸盐转运蛋白的膜蛋白。为了了解这些转运蛋白的功能， 我们从E.大肠杆菌，并进一步证明，令人惊讶的是， NarK是一种硝酸盐/亚硝酸盐交换剂。尽管我们取得了进展，但详细的分子机制 硝酸盐/亚硝酸盐易位在很大程度上仍然未知。在本建议中，我们旨在通过以下方式填补知识空白：1） 了解底物选择性和构象灵活性使用定向诱变的NarK，以便更好地 在分子水平上了解NarK的功能; 2）获得NarK的高分辨率结构， 以前未观察到的构象，因此我们可以重建NarK的完整运输循环; 3）探索 人硝酸盐转运体唾液酸蛋白的结构-功能关系，因此我们将了解其相似性， 不同物种硝酸盐转运蛋白的差异。总体而言，在完成提案后，我们 期望扩大我们对硝酸盐/亚硝酸盐运输的一般理解，并阐明其关键作用 硝酸盐转运蛋白（包括真核和原核）在硝酸盐/亚硝酸盐循环中的作用。知识 获得这里将促进潜在的药物开发有关的细菌硝酸盐转运蛋白和人唾液酸。

Project Summary Recent studies suggest that nitrate and nitrite molecules have profound beneficial effects to human health, though they were thought to be cancerous since 1970s. To maximize their pharmaceutical potential, we need to first understand how nitrate and nitrite circulate in humans. Such circulation depends on two critical events: active accumulation of nitrate mediated by sialin transporters in salivary glands, as well as nitrate uptake and nitrite secretion by commensal bacteria in the mouth. Thus, nitrate/nitrite molecules have to cross different cellular membranes multiple times, before being used by humans. These translocation processes are mediated by a group of membrane proteins called nitrate transporters. To understand how these transporters function, we solved high-resolution crystal structures of NarK from E. coli, and further demonstrated that, surprisingly, NarK is a nitrate/nitrite exchanger. Despite the progress we made, the detailed molecular mechanisms of nitrate/nitrite translocation are still largely unknown. In this proposal, we aim to fill the knowledge gap by: 1) understand substrate selectivity and conformational flexibility using directed-mutagenesis of NarK, so to better understand the function of NarK at the molecular level; 2) obtain high-resolution structures of NarK in previously unobserved conformation, so we can reconstruct the complete transport cycle of NarK; 3) explore the structure-function relationship of human nitrate transporter sialin, so we will understand the similarities and differences among nitrate transporters from diverse species. Overall, upon completion of the proposal, we expect to expand our general understanding of the nitrate/nitrite transport, and shed light on the crucial roles that nitrate transporters (both eukaryotic and prokaryotic) play in nitrate/nitrite circulation. The knowledge gained here will facilitate potential drug development related to bacterial nitrate transporters and human sialin.