Structure-Function Analysis of Leishmania MIT

利什曼原虫 MIT 的结构功能分析

• 批准号: 7046720
• 负责人: Andreas Georg Seyfang
• 金额: $ 14.16万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046720
• 关键词: Leishmania; Xenopus oocyte; chimeric proteins; cysteine; enzyme substrate; genetic mapping; inositol; membrane permeability; membrane transport proteins; permease; polymerase chain reaction; protein isoforms; protein structure function; protozoal genetics; radiotracer; site directed mutagenesis; transfection

DESCRIPTION (provided by applicant): Leishmania parasites are opportunistic protozoan flagellates that are the causative agents of devastating and often fatal diseases in much of the tropical and subtropical world. An increasing problem is the occurrence of Leishmania/HIV co-infection in immunocompromised individuals where persistent and previously asymptomatic parasites develop leishmaniasis after the outbreak of AIDS. In these protozoan flagellates, myo-inositol plays an especially important role as the precursor for GPI-anchored protective and/or immunomodulatory surface molecules, which are several orders of magnitude more abundant on the surface of these parasites than in the mammalian host. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway. For inositol salvage, Leishmania donovani has an active myo-inositol/H+ transporter (MIT) that is driven by a proton-electrochemical gradient across the parasite membrane. Moreover, the proton-coupled Leishmania MIT is functionally and structurally unrelated to the human sodium-coupled myo-inositol transporters (SMIT1 and 2) in the intestine and kidney. Many of the transporters in Leishmania and other protozoan parasites are thought to function as proton-coupled active transporters, but these carriers are in general not well characterized at the molecular level. Leishmania MIT has an exceptionally high substrate specificity, in contrast to the human inositol transporters, and the C-2, C-3, and C-5 hydroxyl groups of myoinositol are critical for substrate recognition by the Leishmania permease. Three specific aims will investigate the structure-function relationship of the L donovani MIT as a promising target for delivery of cytotoxic inositol analogues, and as a model active transporter in these early protozoan eukaryotes. (i) In the first specific aim chimeras between MIT and the structurally related E. coli xylose/H+ symporter (which does not transport myo-inositol) will be generated to investigate the domain(s) in MIT that are responsible for substrate selectivity. (ii) In the second specific aim part of the substrate permeation pathway of MIT will be mapped by cysteine scanning mutagenesis of transmembrane domain 1 (TM1) that contains the functionally essential residues Asp19. These experiments will test the hypothesis that TM1 forms part of the substrate permeation pore that allows active and selective myo-inositol transport across the parasite membrane. Hence the first two specific aims will probe the "active site" of this permease that allows inositol recognition and subsequent transport across the plasma membrane, (iii) MIT belongs to a large sugar transporter superfamily of membrane transporters with 12 transmembrane domains that are thought to have evolved through gene duplication of an ancestral 6-transmembranedomain transporter. Hence the final specific aim will test the hypothesis that either the MIT N-terminal or C-terminal 6 transmembrane segments (N6 or C6, respectively) are sufficient for transport function, similar to an ancestral 6- transmembrane-domain transporter. Functional characterization of the MIT half-transporters N6 or C6 will be performed in the Xenopus oocyte expression system and in an MIT-less L. donovani strain that was recently developed in the laboratory, following transfection with each MIT half-transporter individually, or with N6-N6 and C6-C6 tandem repeat chimera constructs.

描述（由申请人提供）：利什曼原虫寄生虫是机会性原生动物鞭毛虫，是热带和亚热带世界大部分地区毁灭性和致命性疾病的病原体。一个日益严重的问题是利什曼原虫/HIV合并感染在免疫功能低下的个体中的发生，其中持续的和先前无症状的寄生虫在AIDS爆发后发展为利什曼病。在这些原生动物鞭毛虫中，肌醇作为GPI锚定的保护性和/或免疫调节性表面分子的前体起着特别重要的作用，这些分子在这些寄生虫的表面上比在哺乳动物宿主中多几个数量级。此外，肌醇在磷脂酰肌醇信号转导途径中起重要作用。对于肌醇补救，杜氏利什曼原虫具有活性肌醇/H+转运体（MIT），其由跨寄生虫膜的质子电化学梯度驱动。此外，质子偶联利什曼原虫MIT在功能和结构上与肠和肾中的人钠偶联肌醇转运蛋白（SMIT 1和2）无关。利什曼原虫和其他原生动物寄生虫中的许多转运蛋白被认为具有质子偶联活性转运蛋白的功能，但这些载体通常在分子水平上没有很好的表征。与人类肌醇转运蛋白相比，利什曼原虫MIT具有非常高的底物特异性，并且肌醇的C-2、C-3和C-5羟基对于利什曼原虫通透酶的底物识别至关重要。三个具体的目标将调查的结构-功能关系的L donovani MIT作为一个有前途的目标提供细胞毒性肌醇类似物，并作为一个模型，在这些早期的原生动物真核生物的主动转运。(i)在第一个特定的目的嵌合体之间的MIT和结构相关的E。大肠杆菌木糖/H+同向转运体（其不转运肌醇）将被产生以研究MIT中负责底物选择性的结构域。(ii)在第二个具体目标中，将通过半胱氨酸扫描诱变包含功能必需残基Asp 19的跨膜结构域1（TM 1）来定位MIT的底物渗透途径的一部分。这些实验将测试的假设，即TM 1形成的基板渗透孔，允许积极和选择性的肌醇运输通过寄生虫膜的一部分。因此，前两个具体的目标将探测的“活性位点”的这种通透酶，允许肌醇识别和随后的跨质膜运输，（iii）MIT属于一个大的糖转运蛋白超家族的膜转运蛋白与12个跨膜结构域，被认为是通过基因复制的祖先6-transmembranedomain转运蛋白进化。因此，最终的具体目标将测试MIT N-末端或C-末端6跨膜区段（分别为N6或C6）足以用于转运功能的假设，类似于祖先6跨膜结构域转运蛋白。MIT半转运蛋白N6或C6的功能表征将在非洲爪蟾卵母细胞表达系统和无MIT的L. donovani菌株，在用每种MIT半转运蛋白单独转染或用N6-N6和C6-C6串联重复嵌合体构建体转染后。