Calcium-Binding Human Centrosome Proteins and Complexes

翻译后修饰和钙结合是中心体复制和 分离。这些过程在健康组织中受到调节，在疾病状态下是有缺陷的，例如 癌症。异常，例如扩增的和多个中心体，经常在人类乳房中观察到， 结直肠癌和其他癌症。 Centrin是一种EF-Hand蛋白，在中心体中既发挥结构作用又发挥调节作用。这 钙结合蛋白在低钙水平下与一种名为Sfi1的新型1242氨基酸蛋白相互作用， 它包含多达23个中心素结合位点。生物物理、结构和动力学耦合分析 中心素/SfM复合体是理解其生物学功能的基础。 在目标I中，我们将确定与形成中央蛋白-丝裂原复合体有关的相互作用 观察它们在Sfi1的动态收缩和伸长过程中的变化。我们的实验将 解决这种复杂相互作用的分子基础下的关键问题。我们将研究这一过程 的钙结合和磷酸化，并将它们与触发 Sfi1中的收缩和伸长。在目标2中，我们将确定Sfi1的结构并测量其动力学 它的收缩和伸长。我们预计Sfi1的结构特征将使我们深入了解 该蛋白的结构-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 原子核。随着拟议项目的完成，我们预计可以达到我们的短期目标： 在分子水平上描述复合体上中心素和Sfi1的构象变化 队形。我们希望明确Sfi1收缩的分子机制和促进 这件事。此外，对中心素的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被 到目前为止，从结构上解决了问题。 这些结果的意义在于我们对细胞的调节和结构方面的理解 在中心体水平上的分裂。我们的发现将提供关于 中心体蛋白的功能和帮助确定它们的生物活性。

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.