Control of Executioner Caspases with an Allosteric Switch

半胱天冬酶是控制凋亡性细胞死亡的半胱氨酸蛋白酶。如果半胱天冬酶能够被激活， 细胞死亡;相反，抑制半胱天冬酶可以防止心脏病发作和中风等疾病中的细胞死亡。 因此，人们对半胱天冬酶作为药物靶点产生了极大的兴趣。这种兴趣在以下情况下得到了加强： 半胱天冬酶-6被发现在神经变性疾病中起中心作用。不幸的是，到目前为止，没有 半胱天冬酶导向疗法已经上市，主要是因为工作集中在靶向活性 位点，这是该家族最重叠和保守的区域。越来越清楚的是， 每一种半胱天冬酶都以独特而复杂的方式进行调节，因此， 半胱天冬酶特异性抑制可以通过利用变构位点来实现。为了靶向特定的半胱天冬酶或 由于caspase是一组变构的caspase，因此了解单个caspase之间的差异至关重要 以及半胱天冬酶家族中亚组之间的相似性。因此，本项目的目标是了解如何 磷酸化和锌有助于半胱天冬酶活性的调节。理解以下角色 磷酸化或锌结合单独提供了关于天然调节过程的关键信息， 凋亡半胱天冬酶的每个成员。这些地点一起突出了关键的敏感区域， caspase功能的策略控制。 半胱天冬酶被广泛磷酸化。大多数磷酸化事件导致 caspase功能我们的第一种方法使用我们已经开发的结构分析方法， 半胱天冬酶的磷酸模拟物和磷酸化形式。这些结构揭示了机制， 其磷酸化阻止胱天蛋白酶活性并且还鉴定构象控制的关键区域， 其是功能性变构位点。其次，锌可以抑制多种半胱天冬酶， 与细胞凋亡和老年痴呆症有关我们用反常的x射线衍射 实验来鉴定和表征半胱天冬酶中的新锌结合位点。这两种方法： 磷酸化和锌结合以前帮助我们鉴定半胱天冬酶中新的变构位点。通过 系统地应用这些方法，我们可以全面地绘制所使用的变构位点， 以及仅在一种特定胱天蛋白酶上发现的独特位点。我们 方法的目的是提供变构控制的分子细节，以及评估 这些机制的生物学意义。磷酸化caspase变构的比较图谱 和锌结合，我们正在产生的将使我们能够选择最合适的调控位点， 半胱天冬酶功能的最佳控制和涉及半胱天冬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.