Structural characterization of MCE mediated lipid transport across the Mycobacterial cell envelope

项目摘要/摘要： 结核分枝杆菌(Mtb)的细胞膜起着重要的保护作用。 常见的抗菌剂和宿主细胞防御，但也是结核分枝杆菌必须通过的屏障 衍生营养素。MCE蛋白家族(最初与哺乳动物细胞进入有关)几乎普遍存在于 双膜细菌和MCE蛋白现在被认为是促进脂质的转运蛋白 内膜和外膜之间的运动。虽然Mtb MCE蛋白对毒力至关重要，但它们的 结核分枝杆菌的作用机制尚不清楚。由于结核分枝杆菌不同的细胞膜，结核分枝杆菌MCE蛋白可能采用独特的 以建筑为中介的交通。此外，在整个结核分枝杆菌感染过程中，MCE转运体是 被认为是由MCE转录调节因子(MceRs)差异表达的。这表明 分枝杆菌的环境感应反过来又改变了mce操纵子的转录。利用结构生物学， 生物化学和遗传互补分析，我将提供对结核分枝杆菌结构的第一次一瞥 并阐明MceRs如何在分子水平上与其操纵子序列相互作用 规范MCE的运作。为此，我提出了以下目标：(目标1)Mce4脂类的结构测定 污垢分枝杆菌转运系统及(AIM 2)结构特征和DNA结合 MceRs的特异性。我的工作将让我们对污垢分枝杆菌的结构有一个初步的了解。 转运蛋白系统，以及mCER介导的mce操纵子调控的分子机制。 可能有助于阐明结核分枝杆菌细胞膜的维持机制，为临床治疗提供新的靶点 治疗学。

PROJECT SUMMARY / ABSTRACT: The cell envelope of Mycobacterium tuberculosis (Mtb) serves an important role as protection against common antimicrobials and host cell defenses, but also a barrier through which Mtb must transport host derived nutrients. The MCE protein family (originally implicated in Mammalian Cell Entry) is nearly ubiquitous in double-membraned bacteria, and MCE proteins are now believed to act as transporters that facilitate lipid movement between the inner and outer membranes. While Mtb MCE proteins are critical for virulence, their mechanism in Mtb remains unclear. Due to Mtb’s distinct cell envelope Mtb MCE proteins likely adopt a unique architecture to mediate transport. In addition, throughout the course of Mtb infection MCE transporters are believed to be differentially expressed by MCE transcriptional regulators (MceRs). This suggests that environmental sensing in Mycobacteria in turn alters transcription of the mce operons. Using structural biology, biochemistry, and genetic complementation assays, I will provide the first glimpse into the architecture of Mtb Mce protein systems and elucidate how MceRs interact with their operator sequences at the molecular level to regulate mce operons. To do so I propose the following aims: (Aim 1) structure determination of the Mce4 lipid transport system from Mycobacterium smegmatis and (Aim 2) structural characterization and DNA-binding specificity of MceRs. My work will provide the first glimpse at the architecture of the M. smegmatis Mce4 transporter system, as well as the molecular mechanisms of MceR mediated mce operon regulation, which may shed light on the mechanisms of cell envelope maintenance in Mtb and provide a novel target for therapeutics.