Structure-Function Analysis of Leishmania MIT

描述(申请人提供)：利什曼原虫寄生虫是机会主义的原生动物鞭毛虫，在热带和亚热带世界的大部分地区是毁灭性疾病的病原体，通常是致命的。一个日益严重的问题是，在免疫功能受损的人中出现利什曼病/艾滋病毒混合感染，在艾滋病暴发后，持续的和以前没有症状的寄生虫会发展成利什曼病。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比哺乳动物宿主的表面丰富几个数量级。此外，肌醇在磷脂酰肌醇信号转导途径中起着重要作用。对于肌醇回收，杜氏利什曼原虫有一个活性的肌醇/H+转运蛋白(MIT)，该转运蛋白是由跨越寄生虫膜的质子-电化学梯度驱动的。此外，质子偶联利什曼原虫MIT在功能和结构上与人类肠道和肾脏的钠偶联肌醇转运体(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蛋白被认为是质子偶联的活性转运蛋白，但这些载体通常在分子水平上没有很好的特征。与人类肌醇转运蛋白相比，利什曼原虫MIT具有极高的底物特异性，肌醇的C-2、C-3和C-5羟基对利什曼原虫渗透酶识别底物至关重要。三个特定的目标将研究L多诺瓦尼MIT作为细胞毒性肌醇类似物递送的有前景的靶标，以及作为这些早期原生动物真核生物中的模型活性转运蛋白的结构与功能的关系。(I)在第一个特定目标中，将在麻省理工学院和结构上相关的大肠杆菌木糖/H+共转运蛋白(不运输肌醇)之间产生嵌合体，以研究麻省理工学院中负责底物选择性的结构域(S)。(Ii)在第二个特定目标中，MIT底物渗透途径的一部分将通过包含功能必需残基Asp19的跨膜结构域1(TM1)的半胱氨酸扫描突变来定位。这些实验将检验这样一种假设，即TM1形成底物渗透孔的一部分，允许肌醇通过寄生虫膜进行活性和选择性的运输。因此，前两个特定的目的将探索这种渗透酶的“活性部位”，它允许肌醇识别和随后的跨质膜转运；(Iii)MIT属于一个大型的糖转运蛋白超家族，膜转运蛋白具有12个跨膜结构域，被认为是通过祖先的6-跨膜转运蛋白的基因复制而进化的。因此，最终的特定目标将检验这样的假设，即MIT的N-末端或C-末端6跨膜片段(分别为N6或C6)具有足够的转运功能，类似于祖先的6-跨膜域转运蛋白。MIT半转运体N6或C6的功能鉴定将在非洲爪哇卵母细胞表达系统中进行，并在实验室最近培育的无MIT的多诺瓦尼乳杆菌株中进行，分别与每个MIT半转运体或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.