Control of Executioner Caspases with an Allosteric Switch

半胱氨酸蛋白酶是一种控制细胞凋亡的半胱氨酸蛋白酶。如果半胱氨酸天冬氨酸酶被激活，癌症 细胞死亡；相反，抑制半胱氨酸酶可以防止心脏病发作和中风等疾病中的细胞死亡。 因此，人们对半胱氨酸天冬氨酸酶作为药物靶标产生了浓厚的兴趣。这种兴趣在以下情况下得到加强 Caspase-6被发现在神经退行性疾病中发挥核心作用。不幸的是，到目前为止，没有 市场上有caspase导向的疗法，主要是因为研究的重点是针对活跃的 这是该家族中重叠最多、最保守的区域。越来越清楚的是， 每个caspase都以一种独特而复杂的方式进行调控，因此实现 Caspase的特异性抑制可能是通过利用变构位点来实现的。为了针对特定的caspase或 一组caspase变构，了解单个caspase之间的差异是至关重要的 以及半胱氨酸天冬氨酸酶家族亚群之间的相似性。因此，该项目的目标是了解如何 磷酸化和锌参与了caspase活性的调节。了解企业的角色 磷酸化或锌结合本身就提供了有关自然调节过程的关键信息 凋亡的半胱氨酸天冬氨酸酶的每个成员。这些网站一起突出了关键的敏感区域，这些区域允许 Caspase功能的战略控制。 半胱氨酸氨基转移酶被广泛的磷酸化。大多数磷酸化事件都会导致蛋白失活 Caspase功能。我们的第一种方法使用我们开发的方法来进行结构分析 半胱氨酸天冬氨酸酶的拟磷酸和磷酸化版本。这些结构通过以下方式揭示了这种机制 其中的磷酸化可以阻止caspase的活性，也可以识别构象控制的关键区域， 它们是功能性的变构位点。其次，锌可以抑制各种半胱氨酸天冬氨酸酶，这也是 与细胞凋亡和阿尔茨海默病有关。我们用的是反常的x射线衍射 鉴定和表征半胱氨酸天冬氨酸酶中新的锌结合位点的实验。这两种方法都是： 磷酸化和锌结合帮助我们在半胱氨酸酶中发现了新的变构位点。通过 系统地应用这些方法，我们可以全面地绘制出所使用的变构位置 跨caspase家族以及仅在一个特定caspase上发现的唯一位点。我们的 设计的方法是提供变构控制的分子细节以及评估 这些机制的生物学相关性。半胱氨酸天冬氨酸酶变构的磷酸化比较图谱 我们正在产生的锌结合将使我们能够选择最合适的调控位点 对半胱氨酸天冬氨酸酶功能的最佳控制和对涉及半胱氨酸酶的疾病的有效治疗。

Caspases are the cysteine proteases that control apoptotic cell death. If caspases can be activated, cancer cells die; conversely inhibiting caspases could prevent cell death in diseases like heart attack and stroke. Thus there has been significant interest in caspases as drug targets. This interest was heightened when caspase-6 was discovered to play a central role in neurodegenerative diseases. Unfortunately, to date, no caspase-directed therapies are on the market, primarily because work has focused on targeting the active site, which is the most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is regulated in a unique and complex manner so the most promising avenue for achieving caspase-specific inhibition may be by harnessing allosteric sites. In order to target a specific caspase or group of caspases allosterically, it is essential to understand the differences between individual caspases and the similarities within subgroups in the caspase family. Thus, the goal of this project is understand how phosphorylation and zinc contribute to regulation of caspase activity. Understanding the roles of phosphorylation or zinc binding alone provides critical information about natural regulatory processes for each member of the apoptotic caspases. Together these sites highlight key sensitive regions that allow strategic control of caspase function. Caspases are extensively phosphorylated. Most phosphorylation events lead to inactivation of caspase function. Our first approach uses methods we have developed for structural analysis of phosphomimetic and phosphorylated versions of caspases. These structures uncover the mechanism by which phosphorylation prevents caspase activity and also identify key regions of conformational control, which are functional allosteric sites. Second, various caspases can be inhibited by zinc, which has also been linked to apoptosis and Alzheimer's Disease. We are applying anomalous x-ray diffraction experiments to identify and characterize novel zinc-binding sites in caspases. Both of these approaches: phosphorylation and zinc-binding have helped us previously to identify new allosteric sites in caspases. By systematically applying these approaches, we can comprehensively map allosteric sites that are used across the caspase family as well as unique sites that are found only on one particular caspase. Our approaches are designed to provide the molecular details of allosteric control as well as assess the biological relevance of these mechanisms. The comparative map of caspase allostery by phosphorylation and zinc binding that we are generating will enable us to select the most appropriate regulatory sites for optimal control of caspase function and for effective treatment of diseases that involve caspases.