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Mechanism, proton co-transport, and directionality of the formate-nitrite transporter family (FNT)

甲酸盐-亚硝酸盐转运蛋白家族 (FNT) 的机制、质子协同转运和方向性

基本信息

批准号：
200682196
负责人：
Professor Dr. Eric Beitz
金额：
--
依托单位：
Pharmazeutisches Institut
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/200682196?language=en
关键词：
Mechanism proton co transport directionality

项目摘要

The main focus of the first project phase was on the cell-free production and characterization of difficult membrane proteins. Our work on the putative formate-nitrite-transporter (PfFNT) from malaria parasites led to an unexpected breakthrough in the malaria research field. PfFNT has turned into the currently most important candidate as the long-sought lactate-transporter of the parasites und represents a highly promising novel drug target. The work programme of this submission has been adapted according to the recommendations of the reviewers, i.e. removal of the uncertain protein crystallization and shifting of the focus on the study of the basic, largely unknown mechanisms of the formate-nitrite-transporter family with respect to anion transport, proton co-transport, and directionality. We will clone two new FNT proteins from Bacillus thuringiensis (BtFNT) and Entamoeba histolytica (EhFNT), biochemically characterize and compare their properties to established bacterial and plasmodial FNTs. This selection is due to two reasons: 1. B. thuringiensis is closely related to extremely pathogenic Bacillus species, such as the etiologic agent of anthrax, and E. histolytica causes amoebiasis and produces acetate as a metabolic end product, which may be expelled by EhFNT. Both FNTs, thus, are of therapeutic interest. 2. BtFNT and EhFNT exhibit an exchange to glutamine or asparagine, respectively, of a highly conserved histidine within the transport channel. The histidine is central in the current models on the FNT transport mechanism, which postulate protonation of a passing anion by a proton relay comprising the mentioned histidine plus an equally conserved threonine and a fixed water molecule. The amides glutamine and asparagine can neither act as proton acceptors or donors. Apparently, nature has evolved variations, which we want to exploit to gain detailed insight into the protonation mechanism of the FNTs, into proton co-transport, and directionality. We will generate mutants of the critical histidine and threonine residues, use and establish assay systems based on yeast and proteoliposomes, visualize proton co-transport by fluorescent dyes, and set up membrane systems in which the orientation of the FNT proteins is uniform or in which we will selectively inhibit transport in one direction in order to analyze directionality.

第一个项目阶段的主要重点是无细胞生产和表征困难的膜蛋白。我们对疟疾寄生虫推测的甲酸盐-亚硝酸盐转运体(PfFNT)的研究工作在疟疾研究领域取得了意想不到的突破。PfFNT已成为目前最重要的候选药物，因为长期寻找的寄生虫乳酸转运蛋白和代表着一个非常有前途的新药物靶点。根据评审者的建议调整了本呈件的工作方案，即消除不确定的蛋白质结晶，并将重点转移到研究甲酸盐-亚硝酸盐-转运体家族关于阴离子转运、质子共转运和方向性的基本的、基本上未知的机制上。我们将从苏云金芽孢杆菌(BtFNT)和溶组织内阿米巴(EhFNT)中克隆两个新的FNT蛋白，对它们的生化特征进行分析，并将它们的性质与已有的细菌和原虫FNT进行比较。这种选择是由于两个原因：1.苏云金芽孢杆菌与高致病性的芽孢杆菌密切相关，如炭疽的病原体，以及组织溶解埃希氏菌引起阿米巴病并产生乙酸酯作为代谢终产物，可被EhFNT排出。因此，这两种FNT都具有治疗价值。2.BtFNT和EhFNT在转运通道内分别与谷氨酰胺或天冬酰胺交换高度保守的组氨酸。组氨酸在当前关于FNT运输机制的模型中是核心的，这些模型假设通过质子继电器将经过的阴离子质子化，该质子继电器包括所提到的组氨酸加上同样保守的苏氨酸和固定的水分子。氨基谷氨酰胺和天冬酰胺既不能作为质子受体，也不能作为质子供体。显然，自然界已经进化了变异，我们想要利用这些变异来详细了解fnts的质子化机制，到质子共运输和方向性。我们将产生关键组氨酸和苏氨酸残基的突变体，使用和建立基于酵母和蛋白脂质体的检测系统，可视化荧光染料的质子共运输，并建立膜系统，其中FNT蛋白的取向是一致的，或者我们将选择性地抑制一个方向的运输，以便分析方向性。