Structural studies on phospho-regulation of the TACC3 / ChTOG protein complex in mitotic spindle assembly

通过显微镜观察，细胞在进入有丝分裂时经历了一个壮观的转变，有丝分裂是它们分裂前的存在阶段。有丝分裂纺锤体的形成，细胞在哪里？S的微管纤维网络完全重新排列，从两端延伸到中心的染色体，这是特别引人注目的。这个纺锤体是一种分子机器，它确保细胞？S染色体精确地分布在它的两个子细胞之间。纺锤体工作中的错误是已知的癌症的驱动力，也是先天性脑部疾病的原因。有几个控制机制确保有丝分裂纺锤体正常组装正确。在组装的早期阶段，两种叫做TACC3和ChTOG的蛋白质促进微管的稳定性，从而促进组装。当TACC3被磷酸基团修饰时，这些蛋白质更有效：一个磷原子和三个氧原子通常被细胞用来改变蛋白质的活性。在TACC3的例子中，加入磷酸的蛋白质被称为Aurora-A。因此纺锤体组装是由Aurora-A的活性控制的，而Aurora-A本身受细胞内外事件的影响，由许多其他蛋白质控制。我们建议在原子水平上研究磷酸基团如何影响TACC3/ChTOG伙伴关系的有效性。这是如何起作用的，目前是一个谜，因为磷酸盐只有四个原子大，但它改变了TACC3/ChTOG的活性，总共有数万个原子。我们将使用电子显微镜，一种可以让我们直接看到蛋白质形状的技术，来研究TACC3在磷酸化过程中的变化，以及对ChTOG的影响。我们还将使用x射线晶体学来确定蛋白质中每个原子的位置，并绘制TACC3和ChTOG合作的原子图。这些信息将使我们能够对TACC3/ChTOG伙伴关系如何工作以及磷酸化如何增强其有效性的细节做出假设。我们将使用我们的蛋白质结构模型来设计对TACC3和ChTOG的细微修改，以在培养的人类细胞中测试这一假设。过量的TACC3、ChTOG或Aurora-A与癌症有关，TACC3和Aurora-A在大脑发育中也很重要。这些研究将为未来的研究提供动力，以了解这些蛋白质在人类疾病中的作用。

Viewed through a microscope, cells undergo a spectacular transformation as they enter mitosis, the phase of their existence just before they divide. The formation of the mitotic spindle, where the cell?s network of microtubule fibres is completely rearranged to span from either end to the chromosomes at the centre, is particularly striking. This spindle is a molecular machine that ensures the cell?s chromosomes are accurately distributed between its two daughter cells. Errors in the workings of the spindle are a known driving force of cancer and are also responsible for a congenital brain disease. Several control mechanisms ensure the mitotic spindle is normally assembled correctly. At an early stage in assembly, two proteins called TACC3 and ChTOG promote microtubule stability and hence promote assembly. These proteins are more effective when TACC3 is modified by a phosphate group: one phosphorous atom and three oxygen atoms that is commonly used by cells to alter the activity of their proteins. In the case of TACC3, the protein that adds the phosphate is called Aurora-A. Spindle assembly is thus controlled by the activity of Aurora-A, which is itself controlled by many other proteins under the influence of events within and outside the cell. We propose to investigate how the phosphate group influences the effectiveness of the TACC3/ChTOG partnership at the level of atoms. How this works is currently a mystery as the phosphate is only four atoms big, and yet it changes the activity of TACC3/ChTOG which total tens of thousands of atoms. We will use electron microscopy, a technique that allows us to see directly the shapes of proteins, to study the changes in TACC3 upon phosphorylation, and the effect on ChTOG. We will also use X-ray crystallography to determine the location of every atom within the proteins and to map the atoms by which TACC3 and ChTOG cooperate. This information will allow us to make a hypothesis for the details of how the TACC3/ChTOG partnership works and how phosphorylation enhances their effectiveness. We will use our protein structure models to design subtle modifications to TACC3 and ChTOG to test this hypothesis in human cells grown in culture. An overabundance of TACC3, ChTOG or Aurora-A have been linked with cancer, and TACC3 and Aurora-A are also important in brain development. These studies will provide the impetus for future investigations to understand the role of these proteins in human disease.