LISTERIA USES HOST CELL ACTIN TO SPREAD CELL TO CELL

李斯特菌和志贺氏菌劫持宿主的肌动蛋白细胞骨架 细胞，并使用它在细胞之间传播。A幕，李斯特氏菌 表面蛋白是唯一需要的细菌蛋白质 肌动蛋白马达的组装，它直接与Vasp相互作用， 一种宿主细胞骨架蛋白。这种交互至少需要 在ActA中发现的4个串联Pro重复基序之一。 Profilin，一种肌动蛋白结合蛋白，具有几种有效的 对肌动蛋白组装的影响，直接与Vasp结合。这 相互作用由不同的串联脯氨酸重复基序介导 在Vasp上发现，并可被这些Pro基序阻止 活体化验。A幕、Profilin和Vasp在 细菌表面与生长的肌动蛋白之间的界面 尾巴。 该提案旨在确定机制，通过这些机制 蛋白质：蛋白质相互作用使李斯特氏菌和志贺氏菌能够 在其表面组装基于肌动蛋白的马达。调查员 建议调查这些相互作用的性质 ActA、Vasp和Profilin通过一系列基因改变 结合细胞生物学研究ActA上的Vasp结合域 和生化分析。这是一个将深化的研究领域 不仅了解特定的致病过程，而且 也是肌动蛋白调控的分子基础 细胞骨架来实现细胞的运动。 有三个具体目标。目的1是研究分子的结合 ActA，细菌表面蛋白，是唯一的细菌 细菌向宿主细胞蛋白质运动所需的蛋白质 VASP(血管扩张剂刺激的磷酸蛋白)，已知 与Acta捆绑在一起，也是运动所必需的。李斯特菌必须 然后，将Vasp吸引到Acta。ActA有四个寡核苷酸序列 由延伸的氨基酸(第一个，DFPPPPTDE和 其余3例相似，1例仅有3个P与I相隔)； 因此，在起始处带有芳香基团并在侧边 带负电荷的残基。这是关键的VASP绑定 Site和所有4个都被认为是在运行，但这一点还没有得到证实。 利用聚合酶链式反应定点突变和细菌表达， Pi建议通过凝胶过滤和分析来研究结合 离心法鉴定主要结构决定因素 以及ActA和Vasp之间的络合物的结合常数。 低聚脯氨酸类似物的制备和研究如下 复合体形成的竞争性抑制物。 目标2结合两个目标：纽蛋白与血管活性肠肽和ActA的结合 在体内发挥作用。他们的想法是李斯特菌必须吸引Vasp 从宿主中的纽蛋白结合。因此，在体外 结合研究将确定Vasp与 纽蛋白VS Acta。多肽类似物抑制的排序 Acta-Vasp和vinculin-Acta的结合也将在 通过显微注射和延时视频显微镜进行活体观察。李斯特菌 Acta突变体将用穿梭载体PMK4和 同源重组与定义的改变 寡核苷酸重复序列。每个突变体的能力 细菌产生肌动蛋白尾巴，在内部移动并从 将研究细胞间的相互作用和在小鼠中引起疾病。 目的3研究Vasp与Profilin的结合以及Vasp与Profilin的结合。 该络合物与ActA的相互作用。我们的想法是，剖析 与Vasp或Vasp-Acta的结合可能改变剖析的能力 增强肌动蛋白单体上的ATP/ADP交换。

Listeria and Shigella hijack the actin cytoskeleton of their host cell and use it to spread from cell to cell. Act A, a Listeria surface protein is the only bacterial protein required for assembly of the actin motor and it interacts directly with VASP, a host cytoskeletal protein. This interaction requires at least one of the 4 tandem proline repeat motifs found in ActA. Profilin, an actin binding protein that has several potent effects on actin assembly, binds directly to VASP. This interaction is mediated by different tandem proline repeat motifs found on VASP, and can be blocked by these proline motifs in in vivo assays. Act A, profilin, and VASP accumulate at the interface between the bacterial surface and the growing actin tail. This proposal aims to identify mechanisms by which these protein:protein interactions enable Listeria and Shigella to assemble actin-based motors at their surfaces. The investigator proposes to investigate the nature of these interactions between ActA, VASP, and profilin through a series of genetic alterations of the VASP binding domain on ActA combined with cell biological and biochemical assays. this is a research area that will deepen understanding not only of particular pathogenic processes, but also of the molecular basis for regulation of the actin cytoskeleton to achieve cell motility. There are three specific aims. Aim 1 is to study the binding of ActA, the bacterial surface protein which is the only bacterial protein required for bacterial motility to a host cell protein VASP (vasodilator-stimulated phosphoprotein), already known to bind to ActA and also necessary for motility. Listeria must then, attract VASP to ActA. ActA has four oligoproline sequences separated by stretches of amino acids, (the first, DFPPPPTDE and the other 3 are similar, one has only 3 P interrupted with I); thus prolines with an aromatic group at the start and flanked by negatively charged residues. This is the critical VASP binding site and all 4 are thought to operate but this is not proven. Using PCR site-direct mutagenesis and bacterial expression, the PI proposes to study binding by gel filtration and analytical centrifugation to identify the primary structural determinants and binding constants for the complex between ActA and VASP. Oligoproline analogues will be prepared and studied as competitive inhibitors of complex formation. Aim 2 combines two aims: the binding of vinculin to VASP and ActA function in vivo. The thought is that Listeria must attract VASP from the vinculin binding in the host. Therefore in vitro binding studies will determine the relative affinity of VASP to vinculin vs. ActA. The rank order of peptide analogue inhibition of ActA-VASP and Vinculin-ActA binding will also be studied in vivo by microinjection and time-lapse video microscopy. Listeria ActA mutants will be prepared with a shuttle vector PMK4 and homologous recombinations made with defined changes in the oligoproline repeat sequences. The ability of each mutant bacterium to generate actin tails, move within and spread from cell to cell and to cause disease in mice will be studied. Aim 3 is to study the binding of VASP to profilin and the interaction of this complex with ActA. The idea is that profilin binding to VASP or VASP-ActA may alter the ability of profilin to enhance ATP/ADP exchange on actin monomers.