Structure and function of the Mad2 checkpoint protein

Mad2 检查点蛋白的结构和功能

• 批准号: 6796625
• 负责人: Xuelian Luo
• 金额: $ 10.61万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6796625
• 关键词: aneuploidy; biological signal transduction; cell cycle; cell cycle proteins; circular dichroism; conformation; fluorescence resonance energy transfer; ligands; mitotic spindle apparatus; neoplasm /cancer genetics; nuclear magnetic resonance spectroscopy; oncoproteins; protein binding; protein folding; protein protein interaction; protein structure function; western blottings

Dr. Xuelian Luo received her Ph.D. and postdoctoral training in protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. She is currently an Instructor in Dr. Jose Rizo-Rey's lab at UT Southwestern Medical Center. Her long-term goal as an independent scientist is to combine structural biology and cell biology to study cancer-related topics such as cell division and signal transduction. During her mentored training period, she will continue to study the structure and function of the Mad2 spindle checkpoint protein. UT Southwestern is a top biomedical institution with excellent reputations in both structural and cancer biology, and thus provides an excellent environment for her training. The Mad2 spindle checkpoint protein ensures the accurate separation of sister-chromatids by inhibiting the ubiquitin ligase activity of the anaphase-promoting complex (APC) until all chromatids achieve bipolar attachment to the mitotic spindle. Mad2 binds to and sequesters Cdc20, an activator of APC, thereby inhibiting APC. Dr. Luo showed that binding of Mad2 to Cdc20 requires a major conformational switch of Mad2. Remarkably, Mad2 undergoes a similarly dramatic structural change upon binding to its upstream regulator, Mad1. She now shows that, in the absence of ligands, apo-Mad2 spontaneously refolds into an "activated" structural state, which resembles the ligand-bound structure of Mad2 with a vacant ligand-binding site. This form of Mad2 is more potent in blocking APC activity. However, the unassisted structural transition of Mad2 occurs with a timescale of hours, suggesting that other checkpoint components, such as Mad1, might facilitate the conformational activation of Mad2 in vivo. Using a combination of biophysical, biochemical, and cell biological techniques, she proposes to investigate the mechanism and the functional consequences of the unusual structural malleability of Mad2. Aim 1 is to characterize the two folded states of Mad2 in the absence of ligands in vitro and in vivo. The focus of Aim 2 is to understand the mechanism by which Mad2 accomplishes the unusually large conformational change in vitro and in vivo. In Aim 3, she will investigate the kinetic folding pathways of Mad2 in vitro and the possibility of chaperone-assisted Mad2 folding in vivo. Malfunction of the spindle checkpoint may contribute to genetic instability and aneuploidy of tumor cells, and several anti-cancer drugs, including Taxol, kill cancer cells by activating this checkpoint. Her proposed studies will facilitate the design of new anti-cancer drugs.

罗雪莲博士获得博士学位。以及通过核磁共振（NMR）光谱测定蛋白质结构的博士后培训。她目前是UT西南医学中心Jose Rizo-Rey博士实验室的讲师。作为一名独立科学家，她的长期目标是将联合收割机结构生物学和细胞生物学结合起来，研究细胞分裂和信号转导等癌症相关课题。在她的指导培训期间，她将继续研究Mad 2纺锤体检查点蛋白的结构和功能。UT Southwestern是一所顶级生物医学机构，在结构和癌症生物学方面享有盛誉，因此为她的培训提供了良好的环境。Mad 2纺锤体检查点蛋白通过抑制后期促进复合物（APC）的遍在蛋白连接酶活性，直到所有染色单体实现双极附着到有丝分裂纺锤体，确保姐妹染色单体的准确分离。Mad 2结合并螯合APC的激活剂Cdc 20，从而抑制APC。罗博士表明，Mad 2与Cdc 20的结合需要Mad 2的重大构象转换。值得注意的是，Mad 2在与其上游调控因子Mad 1结合时发生了类似的戏剧性结构变化。她现在表明，在没有配体的情况下，apo-Mad 2自发地重新折叠成“活化”结构状态，这类似于Mad 2的配体结合结构，具有空的配体结合位点。这种形式的Mad 2在阻断APC活性方面更有效。然而，Mad 2的无辅助结构转变发生在数小时的时间尺度内，这表明其他检查点组分，如Mad 1，可能有助于Mad 2在体内的构象激活。使用生物物理学，生物化学和细胞生物学技术的组合，她建议研究Mad 2不寻常的结构延展性的机制和功能后果。目的1是在体外和体内的配体的情况下，Mad 2的两个折叠状态的特征。目标2的重点是了解机制 Mad 2在体外和体内都能完成异常大的构象变化。在目标3中，她将研究Mad 2在体外的动力学折叠途径和分子伴侣辅助Mad 2在体内折叠的可能性。纺锤体检查点的功能障碍可能导致肿瘤细胞的遗传不稳定性和非整倍性，包括紫杉醇在内的几种抗癌药物通过激活该检查点来杀死癌细胞。她提出的研究将促进新抗癌药物的设计。