Structure-Function Analysis of Leishmania MIT

描述（由申请人提供）：利什曼原虫寄生虫是机会性鞭毛虫原生动物，在热带和亚热带世界的大部分地区是毁灭性和通常致命疾病的病原体。一个日益严重的问题是在免疫功能低下的个体中发生利什曼原虫/艾滋病毒合并感染，在这些个体中，持续存在的和以前无症状的寄生虫在艾滋病暴发后发展为利什曼原虫病。在这些原生鞭毛动物中，肌醇作为gpi锚定的保护和/或免疫调节表面分子的前体起着特别重要的作用，这些表面分子在这些寄生虫表面上比在哺乳动物宿主中丰富几个数量级。此外，肌醇在磷脂酰肌醇信号转导通路中起重要作用。对于肌醇回收，多诺瓦利什曼原虫有一个活跃的肌醇/H+转运体（MIT），由跨越寄生虫膜的质子电化学梯度驱动。此外，质子偶联的利什曼原虫MIT在功能和结构上与肠和肾中的人钠偶联肌醇转运蛋白（SMIT1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体被认为具有质子偶联活性转运体的功能，但这些转运体通常在分子水平上没有很好的表征。与人类肌醇转运体相比，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渗透酶识别底物至关重要。三个特定的目的将研究L donovani MIT作为细胞毒性肌醇类似物递送的有希望的靶点，以及作为这些早期原生动物真核生物的模型活性转运体的结构-功能关系。(i)在第一个特定目标中，将生成MIT与结构相关的大肠杆菌木糖/H+同调体（不运输肌醇）之间的嵌合体，以研究MIT中负责底物选择性的结构域。（ii）在第二个特定目标中，MIT底物渗透途径的一部分将通过半胱氨酸扫描诱变跨膜结构域1 （TM1）来绘制，TM1包含功能必需残基Asp19。这些实验将验证TM1形成底物渗透孔的一部分的假设，该孔允许活跃和选择性的肌醇通过寄生虫膜运输。因此，前两个特定目标将探索这种渗透酶的“活性位点”，它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，具有12个跨膜结构域，被认为是通过祖先的6个跨膜结构域转运蛋白的基因复制进化而来的。因此，最终的具体目标将测试MIT n端或c端6跨膜片段（分别为N6或C6）足以实现运输功能的假设，类似于祖先的6-跨膜域转运蛋白。MIT半转运体N6或C6的功能表征将在爪蟾卵母细胞表达系统和最近在实验室开发的不含MIT的L. donovani菌株中进行，分别转染每个MIT半转运体，或用N6-N6和C6-C6串联重复嵌合体构建。

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.