Control of Executioner Caspases with an Allosteric Switch

项目总结 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点产生了浓厚的兴趣。这种兴趣在以下情况下进一步加剧 Caspase-6被发现在神经退行性变中起核心作用。不幸的是，到目前为止，还没有caspase- 市场上有定向疗法，主要是因为工作主要集中在活跃的部位，即 家族中重叠和保守最多的区域。越来越清楚的是，每一个caspase都是 以一种独特而细微的方式进行调控，因此实现caspase特异性抑制的唯一希望是通过 利用它们的调控，这通常是在变构部位和外生体介导的。为了将目标定位为 对于特定的caspase、caspase组或caspase底物的子集，了解它们之间的差异是至关重要的。 单个caspase之间的引用和caspase亚组内的相似性。因此，我们的长期 该项目的目标一直是定义和利用每个凋亡半胱氨酸酶的独特调控功能。 通过鉴定变构中心和外胚体，我们观察和描述了四个主要的机理 外生体和变构调节的类别。第一个是许多截然不同的监管机构所共有的，即间接监管 破坏协同形成底物结合槽并影响催化的环。基于 我们通过环中断对caspase调节的研究，我们开发了一种更有效的变构抑制剂 也是到目前为止最有选择性的，对caspase-6的偏好是所有其他的500倍 半胱氨酸酶。这种选择性是可以实现的，因为这个新的变构位点只存在于caspase-6中。 鉴于这一成功，我们的目标是研究剩下的三类变构调节。在目标1中，我们 重点研究第二类，鉴定caspase-6及其底物上的外切酶。这种协调一致的分析是可能的 第一次由于我们开发了具有caspase-6活性位点特异性的杂交caspase，但是 Caspase-7的外切点。我们的目标是阻止特定的外生体，并探索其对蛋白质组的影响 基础。我们预计，这种方法将使新的抑制剂的开发，以阻断切割 阿尔茨海默病和亨廷顿病的致病caspase-6底物，如DJ-1、Tau或Huntingtin，但不是其他 底物。在目标2中，我们专注于III类，天然小分子结合。我们最新的发现是ATP 结合到一个孤儿变构空腔，新的方法将使我们能够识别共价和非 从这个位点调控caspase-6的共价天然配体。这一目标意义重大，因为它将提供以下需求- 艾德对半胱氨酸天冬氨酸酶和新陈代谢之间的交集的见解。在目标3中，我们将重点放在第四类，即 影响折叠状态。我们询问了一个caspase-9位点，当它被磷酸化时，会导致 核心。这是所有人类半胱氨酸酶中唯一保守的磷酸化位点。同舟共济 这项工作对增生性和神经退行性疾病都有重要的治疗意义。

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.