Structure and function of the Mad2 checkpoint protein

罗雪莲博士获得了核磁共振波谱测定蛋白质结构的博士和博士后培训。她目前是德克萨斯大学西南医学中心何塞·里佐-雷博士实验室的讲师。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，研究与癌症相关的话题，如细胞分裂和信号转导。在她的指导训练期间，她将继续研究MAD2纺锤体检查点蛋白的结构和功能。德克萨斯大学西南分校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极高的声誉，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，直到所有染色单体都达到两极附着在有丝分裂纺锤体上。MAD2结合并隔离APC的激活剂CDC20，从而抑制APC。罗博士指出，MAD2与Cdc20的结合需要MAD2的主要构象开关。值得注意的是，MAD2在与其上游调控因子Mad1结合后，经历了类似的戏剧性结构变化。她现在证明，在没有配体的情况下，apo-MAD2自发地重新折叠到一种“激活”的结构状态，这类似于MAD2的配体结合结构，但有一个空白的配体结合位点。这种形式的MAD2在阻断APC活性方面更有效。然而，MAD2的非辅助结构转变发生的时间尺度为数小时，这表明其他检查点组件，如MAD1，可能有助于MAD2在体内的构象激活。她建议结合生物物理、生化和细胞生物学技术，研究MAD2异常的结构延展性的机制和功能后果。目的1研究MAD2在体内外无配体情况下的两种折叠状态。目标2的重点是了解这种机制。 通过它，MAD2在体外和体内完成了异常大的构象变化。在目标3中，她将研究MAD2在体外的动力学折叠途径，以及在体内伴侣辅助MAD2折叠的可能性。纺锤体检查点的故障可能导致肿瘤细胞的遗传不稳定和非整倍体，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。她提出的研究将有助于设计新的抗癌药物。

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.