Calcium-Binding Human Centrosome Proteins and Complexes

翻译后修饰和钙离子结合是中心体复制的关键前体， 分居这些过程在健康组织中受到调节，在疾病状态中有缺陷， 癌在人类乳腺中经常观察到异常，如扩增的和多个中心体， 结直肠癌和其他癌症。 中心蛋白是一种在中心体中起结构和调节作用的EF手蛋白。这 钙结合蛋白在低钙水平下与称为Sfi 1的新的1242-氨基酸蛋白相互作用， 其含有多达23个中心蛋白结合位点。耦合生物物理，结构和动态分析 中心蛋白/SfM复合物对于理解其生物学功能至关重要。 在目的I中，我们将确定参与中心蛋白-Sfil复合物形成的相互作用， 观察它们在Sfi 1动态收缩和伸长过程中的变化。我们的实验将 解决这一复杂相互作用的分子基础的关键问题。我们将研究 钙结合和磷酸化的中心蛋白，并将它们与结构变化，触发 Sfi 1的收缩和伸长。在目标2中，我们将确定Sfi 1的结构并测量动力学 它的收缩和伸长。我们希望Sfi 1的结构表征将有助于了解 这种蛋白质的结构-功能关系。 我们的结果将阐明新复制的中心体是如何分离并迁移到细胞的相对侧的。 原子核在建议的工程计划完成后，我们预期可达致短期目标， 在分子水平上描述了在复合物发生时中心蛋白和Sfi 1的构象变化， 阵我们希望能够确定Sfi 1收缩的分子机制以及促进Sfi 1收缩的条件。 大会及其他人此外，中心蛋白磷酸化形式的表征将大大有助于 这种翻译后修饰的结构知识，因为很少有磷酸化的蛋白质已被 到目前为止，在结构上。 这些结果的意义在于我们理解了细胞的调控和结构方面， 分裂在中心体水平。我们的研究结果将提供有关机制的重要信息， 中心体蛋白的功能和帮助确定其生物活性。

Post-translational modification and calcium binding are key pre-requisites for centrosome duplication and separation. These processes are regulated in healthy tissues and are defective in disease states such as cancer. Abnormalities, such as amplified and multiple centrosomes, are often observed in human breast, colorectal, and other cancers. Centrin is an EF-hand protein that plays both structural and regulatory roles in the centrosome. This calcium-binding protein interacts at low calcium levels with a novel 1242-amino acid protein known as Sfi1, which contains up to 23 centrin-binding sites. Coupled biophysical, structural, and dynamic analyses of the centrin/SfM complex are essential to the understanding of its biological function. In Aim I, we will determine the interactions involved in the formation of the centrin-Sfil complex and observe how they change during the dynamic contraction and elongation of Sfi1. Our experiments will address key questions underlying the molecular basis of this complex interaction. We will study the processes of calcium-binding and phosphorylation in centrin and relate them to the structural changes that trigger contraction and elongation in Sfi1. In Aim 2, we will determine the structure of Sfi1 and measure the kinetics of its contraction and elongation. We expect that structural characterizations of Sfi1 will yield insight into the structure-function relationship of this protein. Our results will elucidate how newly duplicated centrosomes separate and migrate to opposite sides of the nucleus. With the completion of the proposed project, we expect to attain our short-term goal of describing at the molecular level the conformational changes that occur in centrin and Sfi1 upon complex formation. We hope to define the molecular mechanism of Sfi1 contraction and the conditions that facilitate this event. In addition, characterization of the phosphorylated form of centrin will contribute greatly to structural knowledge of this post-translational modification, since few phosphorylated proteins have been structurally resolved to date. The significance of these results lies in our understanding of the regulatory and structural aspects of cell division at the centrosome level. Our findings will provide essential information on the mechanisms by which centrosomal proteins function and help define their biological activities.