Structure-Function of the Cytoskeleton

本课题研究血管平滑肌肌动蛋白细胞骨架重构的分子基础。 收缩。它补充了该赠款的其他专注于组织、细胞和大分子的项目 装配。两个肌动蛋白结合蛋白家族，Ena/Vasp和WASP已成为关键的调节因子 细胞骨架重塑，分别在细丝成核和伸长中起着不同的作用。这个 控制这两个过程的分子机制仍然是一个谜。ENA/VASP和WASP在功能上 不同但共享相似的模块化结构。它们都含有多聚-Pro区域，介导结合 Profilin-肌动蛋白，然后是WASP-Homology 2(WH2)类型的G-肌动蛋白结合域。此项目构建 根据重要的初步结果(这里给出)和我们最近确定的各种WH2-结构- 肌动蛋白复合体提出以下两个假设：1)串联WH2s将肌动蛋白亚基沿着 形成肌动蛋白组装的核的丝链(成核步骤)。2)Poly-Pro-WH2模块贡献 通过“加工”Profilin-肌动蛋白复合体以使其结合到带刺的末端来延长细丝 长丝(伸长步骤)。为了检验这些假设，目标1和目标2将剖析结构- Poly-Pro-WH2和Tandem-WH2模块的功能。我们将确定肌动蛋白的晶体结构 通过串联WH2杂化结构组装的微丝以及与Profilin-Actin结合的PolyPro-WH2的微丝 (伸长过程中的过渡态)。补充性解决方案研究将使用分析 超速离心和SAXS/WAXS。各种蛋白质-蛋白质相互作用的生物物理研究 将研究变构效应在这些过程中的作用。AIM 3研究 肌动蛋白结合和支架功能的α-肌动蛋白，一个关键的适配蛋白的血影蛋白家族。 α-肌动蛋白介导质膜整合素与焦点粘连的相互作用 蛋白质Zysin、vinculin和Paladin，进而招募Ena/Vasp到致密斑块。作为一种范例 在这些相互作用中，我们将研究α-肌动蛋白-凝乳素相互作用的结构基础。vbl.使用 SAXS/WAXS，FRET和AU，我们将测试两种流行的F-肌动蛋白结合模型，紧凑型和扩展型， 对于血影蛋白家族成员的肌动蛋白结合域。了解病毒的分子基础 血管肌肉收缩将加速发现治疗心血管疾病的方法。

This project studies the molecular basis for actin cytoskeleton remodeling in vascular smooth muscle contraction. It complements the other projects of this grants that focus on tissues, cells and macromolecular assemblies. Two families of actin-binding proteins, Ena/VASP and WASP, have emerged as key regulators of cytoskeleton remodeling, playing distinct roles in filament nucleation and elongation, respectively. The molecular mechanisms controlling both processes remain a mystery. Ena/VASP and WASP are functionally distinct but share similar modular structures. They both contain poly-Pro regions that mediate the binding of profilin-actin, followed by G-actin binding domains of the WASP-Homology 2 (WH2) type. This project builds upon important preliminary results (presented here) and our recently determined structures of various WH2- actin complexes to propose the following two hypotheses: 1) Tandem WH2s line up actin subunits along a filament strand forming nuclei for actin assembly (nucleation step). 2) The poly-Pro-WH2 module contributes to filament elongation by "processing" profilin-actin complexes for their incorporation onto the barbed end of growing filaments (elongation step). To test these hypotheses, aims 1 and 2 will dissect the structure- function of the poly-Pro-WH2 and tandem-WH2 modules. We will determine the crystal structures of actin minifilaments assembled via tandem WH2 hybrid constructs and that of poly-Pro-WH2 bound to profilin-actin (transition state in elongation). Complementary solution studies will be carried out using analytical ultracentrifugation and SAXS/WAXS. A biophysical study of the various protein-protein interactions involved in nucleation and elongation will investigate the role of allosteric effects in these processes. Aim 3 studies the actin binding and scaffolding functions of alpha-actinin, a key adaptor protein of the spectrin family. Alpha-actinin mediates the interactions between integrins at the plasma membrane and the focal-adhesion proteins zyxin, vinculin, and paladin, which in turn recruit Ena/VASP to dense plaques. As a paradigm of these interactions, we will study the structural basis for the alpha-actinin-zyxin interaction. Using SAXS/WAXS, FRET and AU, we will test the two prevailing F-actin-binding models, compact and extended, for the actin-binding domain of members of the spectrin family. Understanding the molecular basis of vascular muscle contraction will accelerate the discovery of therapies to treat cardiovascular diseases.