Control of Executioner Caspases with an Allosteric Switch

项目摘要 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。这种兴趣进一步增强， 半胱天冬酶-6被发现在神经变性中起中心作用。不幸的是，到目前为止，没有半胱天冬酶- 市场上的定向疗法，主要是因为工作集中在活性部位，这是 家族中最重叠和保守的区域。越来越清楚的是，每一个半胱天冬酶 以独特和微妙的方式进行调节，因此实现caspase特异性抑制的唯一希望是通过 利用它们的调节，这通常是在变构位点和外切位点介导的。为了瞄准一个 具体的caspase，一组caspase，或caspase底物的子集，这是至关重要的，以了解不同的。 个体半胱天冬酶之间的推论和半胱天冬酶亚组内的相似性。因此，我们的长期 项目目标是确定和开发每种凋亡半胱天冬酶的独特调控特征。 通过识别变构位点和外切位点，我们观察并描述了四种主要的机制， 类的exosite和变构调节。第一种是间接的，这是许多不同监管机构所共有的。 破坏协同形成基底结合凹槽并影响催化的环。基于 我们通过环破坏来研究半胱天冬酶的调节，我们开发了一种变构抑制剂， 比任何报道的都多，也是迄今为止最具选择性的，比所有其他的选择性高500倍。 半胱天冬酶这种选择性是可实现的，因为这种新的变构位点仅存在于胱天蛋白酶-6中。 鉴于这一成功，我们的目标是调查其余三类变构调节。目标1： 专注于II类，识别caspase-6及其底物上的外切位点。这种协调一致的分析是可能的 由于我们开发了具有caspase-6活性位点特异性的杂合caspase， caspase-7的外切位点。我们的目标是阻止特定的外切位点，并探索对蛋白质组范围的影响。 基础我们预计，这种方法将使新的抑制剂，阻断切割的发展。 在阿尔茨海默病和亨廷顿病中引起疾病的半胱天冬酶-6底物如DJ-1、Tau或亨廷顿蛋白，但不是其他 印刷受体.在目标2中，我们专注于III类，天然小分子结合。我们最近发现ATP 结合到孤儿变构腔，新的方法将使我们能够识别共价和非共价的， 共价天然配体从该位点调节胱天蛋白酶-6。这一目标意义重大，因为它将提供必要的- 艾德对半胱天冬酶和新陈代谢之间的交叉点有了深入的了解。在目标3中，我们关注第四类， 影响折叠状态。我们询问了一个caspase-9位点，当它磷酸化时， 核心的这是所有人类半胱天冬酶中唯一保守的磷酸化位点。一起 这项工作在增殖性和神经变性疾病中具有重要的治疗意义。

PROJECT SUMMARY Caspases are cysteine proteases that control apoptotic cell death. If caspases are activated, cancer cells die; conversely, inhibiting caspases can prevent cell death in diseases such as heart attack and stroke. Thus, there has been significant interest in caspases as drug targets. This interest heightened further when caspase-6 was discovered to play a central role in neurodegeneration. Unfortunately, to date, no caspase- directed therapies are on the market, primarily because work has focused on 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 nuanced manner, so the only hope for achieving caspase-specific inhibition is by harnessing their regulation, which is usually mediated at allosteric sites and exosites. In order to target a specific caspase, group of caspases, or subset of caspase substrates, it is essential to understand the dif- ferences between individual caspases and the similarities within caspase subgroups. Thus, our long-term project goal has been to define and exploit unique regulatory features for each of the apoptotic caspases. By identifying allosteric sites and exosites, we have observed and described four major mechanistic classes of exosite and allosteric regulation. The first, shared by many disparate regulators, is the indirect disruption of the loops that cooperatively form the substrate-binging groove and impact catalysis. Based on our studies of caspase regulation via loop disruption, we developed an allosteric inhibitor that is more potent than any reported and is also by far the most selective, preferring caspase-6 by 500-fold over all other caspases. This selectivity is achievable because this new allosteric site is present exclusively in caspase-6. Given this success, we aim to investigate the remaining three classes of allosteric regulation. In Aim 1, we focus on class II, identifying exosites on caspase-6 and its substrates. This concerted analysis is possible for the first time due to our development of a hybrid caspase with the active site specificity of caspase-6 but the exosites of caspase-7. We aim to block particular exosites and explore the impact on a proteome-wide basis. We anticipate that this approach will enable the development of new inhibitors that block cleavage of disease-causing caspase-6 substrates like DJ-1,Tau or huntingtin in Alzheimer and Huntington but not other substrates. In Aim 2, we focus on class III, native small molecule binding. Our recent discovery that ATP binds to an orphan allosteric cavity and new methods will allow us to identify both covalent and non- covalent native ligands that regulate caspase-6 from this site. This goal is significant as it will provide need- ed insights into the intersection between caspases and metabolism. In Aim 3, we focus on class IV, which impact the folded state. We interrogate a caspase-9 site that when phosphorylated leads to disassembly of the core. This is the only site of phosphorylation that is conserved among all human caspases. Together this work has significant therapeutic implications in both proliferative and neurodegenerative diseases.