The role of intrinsic disorder in the allosteric regulation of human UGDH

 描述（由申请人提供）：葡萄糖醛酸化通常是导致药物在临床试验期间失败的不利药代动力学或药效学的来源，因此，药物开发迫切需要控制葡萄糖醛酸化的工具。我们的长期目标是开发控制葡萄糖醛酸化的药物。作为实现这一目标的第一步，我们将确定控制人UDP-葡萄糖脱氢酶（hUGDH）的变构机制，该酶产生葡萄糖醛酸化的必需底物。具体而言，hUGDH含有30个残基的固有无序的C-末端（ID-尾），其对于下游代谢物UDP-Xyl的反馈抑制是重要的。我们假设ID-尾和变构“热点”在动态特征的网络内相互作用，以有利于对变构抑制剂具有更高亲和力的hUGDH的构象。我们的假设是基于以下观察：（i）ID-尾和热点控制hUGDH被别构激活或抑制的速率;（ii）ID-尾和热点增加hUGDH被别构激活或抑制的速率。 通过远程机制对变构抑制剂的亲和力;和（iii）ID尾变化 hUGDH的构象系综。所提出的项目的基本原理是，通过了解hUGDH的非活性状态如何被控制而获得的知识有可能转化为使耐药肿瘤对现有的药物敏感的新策略。 抗癌药将通过追求三个具体目标来测试该假设：1）确定非活性状态和活性状态之间的缓慢异构化如何与变构相关联; 2）E250/T253变构热点和hUGDH的固有无序C-末端尾如何有助于反馈抑制;和3）确定ID-尾如何稳定hUGDH的非活性构象。本申请中提出的研究是创新性的，因为它专注于hUGDH的变构抑制作为控制葡萄糖醛酸化的全局机制，并使用我们发现的抑制肽。由于我们的实验室最近才发现内在障碍的作用和控制hUGDH的变构开关的身份，这项研究与以前试图控制葡萄糖醛酸化的尝试不同。这项工作的预期成果是重要的。对hUGDH的非活性状态的详细描述将作为设计一类变构酶的模板，所述变构酶将作为葡萄糖醛酸化的全局调节剂。由于蛋白质组中存在长（>30个残基）的内在无序片段（占所有蛋白质的33%），我们对无序片段对蛋白质结构-功能关系的影响的了解可能会产生广泛的影响。

 DESCRIPTION (provided by applicant): Glucuronidation is often the source of unfavorable pharmacokinetics or pharmacodynamics that lead to the failure of drugs during clinical trials, and as such, there is a critical need in drug development for a tool to control glucuronidation. Our long-term goal is to develop drugs that will control glucuronidation. As a first step toward that goal, we will determine the allosteric mechanism that controls human UDP-Glucose Dehydrogenase (hUGDH), the enzyme that produces the essential substrate for glucuronidation. Specifically, hUGDH contains a 30-residue intrinsically disordered C-terminus (the ID-tail) that is important for feedback inhibition by UDP-Xyl, a downstream metabolite. We hypothesize that the ID-tail and an allosteric 'hotspot' interact within a network of dynamic features to favor a conformation of hUGDH with a higher affinity for the allosteric inhibitor. Our hypothesis is based on the observation that: (i) the ID-tail and the hotspot control the rate at which hUGDH is allosterically activated or inhibited; (ii) the ID-tail and the hotspot increase the affinity for the allosteric inhibitor through a long-range mechanism; and (iii) the ID-tail changes the conformational ensemble of hUGDH. The rationale underlying the proposed project is that the knowledge gained by understanding how the inactive state of hUGDH is controlled has the potential to translate into novel strategies for sensitization of drug resistant tumors to existing cancer drugs. This hypothesis will be tested by pursuing three specific aims: 1) determine how the slow isomerization between the inactive and active states is linked to allostery; 2) how the E250/T253 allosteric hotspot and the intrinsically disordered C-terminal tail of hUGDH contributes to feedback inhibition; and 3) determine how the ID-tail stabilizes the inactive conformation of hUGDH. The research proposed in this application is innovative because it focuses on the allosteric inhibition of hUGDH as a global mechanism for controlling glucuronidation, and uses an inhibitory peptide that we discovered. Since the role of intrinsic disorder and the identity of the allosteric switches that control hUGDH were only recently discovered by our lab, this research is distinct from previous attempts that tried to control glucuronidation. The expected outcomes of this work are significant. A detailed description of the inactive state of hUGDH will act as a template for the design of a class of allosteric inhibitos that will act as global regulators of glucuronidation. Because of the persistence of long (>30 residues) intrinsically disordered segments in the proteome, (33% of all proteins), what we learn about the effect of disordered segments on protein structure-function relationships is likely to have a broad impact.