Structure and function of the Mad2 checkpoint protein

Mad2 检查点蛋白的结构和功能

• 批准号: 7270547
• 负责人: Xuelian Luo
• 金额: $ 14.74万
• 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/7270547
• 关键词: Address; Adopted; Aneuploidy; Antineoplastic Agents; Bacteria; Binding; Binding Sites; Biochemical; Biological; C-terminal; Cancer Biology; Cell division; Cells; Cellular biology; Chromatids; Complex; Defect; Dimerization; Doctor of Philosophy; Employee Strikes; Ensure; Environment; Exhibits; Figs - dietary; Genetic; Goals; Hour; In Vitro; Institution; Kinetics; Ligand Binding; Ligands; Light; Malignant Neoplasms; Medical center; Mentors; Mitosis; Mitotic spindle; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Target; Mutation; N-terminal; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Paclitaxel; Pathway interactions; Peptides; Pliability; Principal Investigator; Proteins; Recombinants; Recruitment Activity; Research; Scientist; Signal Transduction; Sister Chromatid; Structure; Techniques; Training; anaphase-promoting complex; cancer cell; conformer; design; dimer; in vivo; insight; instructor; interdisciplinary approach; killings; monomer; mutant; neoplastic cell; post-doctoral training; prevent; programs; protein structure; structural biology; ubiquitin ligase

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.

罗雪莲博士博士及博士后研究方向为核磁共振（NMR）光谱法测定蛋白质结构。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与癌症相关的课题。在她的指导培训期间，她将继续研究Mad2纺锤体检查点蛋白的结构和功能。UT Southwestern是一所顶尖的生物医学机构，在结构生物学和癌症生物学方面都享有很高的声誉，因此为她的培训提供了良好的环境。Mad2纺锤体检查点蛋白通过抑制后期促进复合体（APC）的泛素连接酶活性，确保姐妹染色单体的准确分离，直到所有染色单体实现双极性附着到有丝分裂纺锤体上。Mad2结合并隔离APC的激活剂Cdc20，从而抑制APC。Luo博士表明，Mad2与Cdc20的结合需要Mad2的一个主要构象开关。值得注意的是，Mad2在与其上游调控因子Mad1结合后也经历了类似的剧烈结构变化。她现在表明，在没有配体的情况下，载脂蛋白Mad2会自发地重新折叠成一种“激活”的结构状态，这种结构类似于带有空配体结合位点的Mad2的配体结合结构。这种形式的Mad2在阻断APC活性方面更有效。然而，Mad2的无辅助结构转变发生在几个小时的时间尺度上，这表明其他检查点成分，如Mad1，可能促进体内Mad2的构象激活。利用生物物理、生化和细胞生物学技术的结合，她建议研究Mad2不寻常的结构延展性的机制和功能后果。目的1是表征Mad2在体内和体外无配体情况下的两种折叠状态。目标2的重点是了解其机制