Human PBGS Quaternary Structure Dynamics, Drugs, and Half-of-the-sites Reactivity

人类 PBGS 四级结构动力学、药物和半位点反应性

• 批准号: 8146896
• 负责人: EILEEN K JAFFE
• 金额: $ 27.21万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8146896
• 关键词: Active Sites; Address; Adjuvant; Adverse drug effect; Adverse effects; Affect; Allosteric Regulation; Amino Acids; Aminolevulinate; Aminolevulinic Acid; Behavior; Binding; Biological; Biological Models; Catalysis; Chlorophyll; Cobalamin; Communication; Coupled; Dermatologic; Diagnostic; Disease; Dissociation; Distant; Effectiveness; Electrophoretic Mobility Shift Assay; Environmental Pollution; Enzymes; Equilibrium; Exhibits; Funding; Gel; Grant; Health; Heme; Human; Human Activities; Individual; Investigation; Ions; Knowledge; Label; Laboratories; Lead Poisoning; Lesion; Levulan; Libraries; Light; Literature; Location; Maintenance; Mediating; Medicine; Metals; Methods; Modality; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Names; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Photochemotherapy; Physiological; Porphobilinogen; Porphobilinogen Synthase; Porphyrias; Porphyrins; Protein Family; Proteins; Public Health; Regulation; Relative (related person); Role; Schiff Bases; Severities; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Symptoms; Techniques; Tetrapyrroles; Therapeutic; Therapeutic Agents; United States; Variant; Zinc; base; cell killing; cofactor; cost; dimer; enzyme activity; fascinate; human disease; improved; inhibitor/antagonist; metalloenzyme; neurotoxic; novel; novel strategies; prevent; protein structure function; public health relevance; response; small molecule; stoichiometry; success

DESCRIPTION (provided by applicant): Human porphobilinogen synthase (PBGS; E.C. 4.2.1.24) catalyzes the ancient and essential formation of the fundamental biological monopyrrole, porphobilinogen in the heme biosynthetic pathway. It is a zinc metalloenzyme and the target enzyme in lead poisoning. Human PBGS participates in a quaternary structure equilibrium that includes high activity octamers, low activity hexamers, and two conformations of a dimer, each of which dictates the stoichiometry of further assembly. Mutations that perturb the quaternary structure equilibrium toward the low activity hexamer are associated with ALAD porphyria, a rare but serious disease state. Photodynamic therapy of dermatologic lesions with the drug Levulan requires PBGS activity. Two distinct aims are proposed. AIM 1 investigates whether drugs and environmental contaminants can modulate the human PBGS quaternary structure equilibrium and thus inhibit or activate enzyme activity. Libraries of approved drugs and known environmental contaminants will be evaluated by a native PAGE mobility shift assay to determine which components can shift the human PBGS quaternary structure equilibrium toward either the low or high activity states. Established methods will be used to validate hits as inhibitors or activators. Inhibitors are molecules that should be avoided in patients with lead poisoning or porphyria; activators may alleviate some symptoms of lead poisoning, serve as a therapeutic for ALAD porphyria, or as adjuvant to optimize the outcome of photodynamic therapy. Coupled with the understanding of how human allelic variation affects the equilibrium of assembly states, these studies introduce a new approach to the application of quaternary structure to personalized medicine. They provide a possible explanation for off-target side effects, and also suggest off-label therapies. AIM 2 uses human PBGS as a model system for understanding the structural basis of half-of-the-sites reactivity (HSR). HSR is similar to allosteric regulation in that binding phenomena at one location in the protein profoundly affect the binding/reactivity behavior at a distant site in the same protein. Intersubunit communication is required for HSR and, in general, the molecular basis for the required communication is poorly understood. Our understanding of human PBGS structure and mechanism allows us to modulate the individual intersubunit interfaces, determine which is responsible for the required communication, and probe individual residues to determine the communication pathway. Quantitative Schiff base trapping will be used as a diagnostic for HSR. We have established that intersubunit interactions that occur when the active site lid is closed are required for maintenance of the active octamer. We propose that substrate/product binding, which is essential for active site lid closure, is therefore also essential for maintenance of the octamer and dictates the requirement for HSR. The proposed studies will improve our understanding of the role of alternate quaternary structure assemblies in human disease and will enhance general knowledge about intersubunit communication essential for HSR and some forms of allostery. PUBLIC HEALTH RELEVANCE: Understanding protein structure-function relationships is essential to understanding the mechanism of action of myriad therapeutic agents. Investigation of porphobilinogen synthase, which participates in a newly discovered regulation mechanism involving quaternary structure dynamics, provides a novel structural basis for understanding the role of alternate protein assemblies in human disease, provides a mechanism for understanding drug side effects, and suggests new therapies. Inactivation of human PBGS is involved in the human conditions of ALAD porphyria and lead poisoning, the latter of which remains a pervasive public health problem in the United States and around the world.

描述（由申请人提供）：人卟啉原合酶（PBGS; E.C. 4.2.1.24）催化基本生物学单胃吡咯，斑膜生物合成途径中基本生物学单胃吡咯的古老而必不可少的形成。它是锌金属酶和铅中毒中的靶酶。人类PBG参与第四纪结构平衡，包括高活动八聚体，低活性六聚体和两个二聚体的构象，每个二聚体都决定了进一步组装的化学计量。扰动四级结构均衡向低活性六聚体的突变与罕见但严重的疾病状态的Alad Porphyria相关。用药物levulan对皮肤病变的光动力疗法需要PBGS活性。提出了两个不同的目标。 AIM 1研究药物和环境污染物是否可以调节人PBG季季结构平衡，从而抑制或激活酶活性。批准的药物和已知环境污染物的库将通过天然页面移动性转移测定法进行评估，以确定哪些组件可以将人类PBG季季结构平衡转移到低活动状态或高活动状态。已建立的方法将用于验证抑制剂或激活剂的命中。抑制剂是铅中毒或卟啉症患者应避免的分子。激活剂可以减轻铅中毒的某些症状，用作Alad Porphyria的治疗性，或者作为优化光动力疗法结果的辅助药。加上对人类等位基因变异如何影响组装状态平衡的理解，这些研究引入了一种新方法，以将第四纪结构应用于个性化医学。它们为脱靶副作用提供了一种可能的解释，还建议使用标签外疗法。 AIM 2使用人类PBG作为模型系统，以理解半位反应性（HSR）的结构基础。 HSR与变构调节相似，因为该蛋白质中一个位置的结合现象深刻影响了同一蛋白质中遥远位点的结合/反应性行为。 hsr需要的亚基间交流，通常，所需通信的分子基础知之甚少。我们对人类PBG的结构和机制的理解使我们能够调节单个的亚基界面，确定哪个负责所需的通信，并探测单个残基以确定通信途径。定量的Schiff基座陷阱将用作HSR的诊断。我们已经确定，在维护活动八聚体时，需要关闭活动位点盖时发生的亚基间相互作用。因此，我们建议底物/产品结合对于维持八聚体的维护以及决定HSR的要求也至关重要。拟议的研究将提高我们对替代四级结构组件在人类疾病中的作用的理解，并将增强对hsr和某些形式的变构的一般知识。 公共卫生相关性：了解蛋白质结构功能关系对于理解众多治疗剂的作用机理至关重要。参与涉及第四纪结构动力学的新发现的调节机制的卟啉原合酶的研究为理解交替蛋白质组件在人类疾病中的作用提供了一种新的结构基础，为了解药物副作用提供了一种机制，并提出了新的疗法。人类PBG的失活参与了Alad Porphyria和铅中毒的人类状况，后者仍然是美国和世界各地普遍存在的公共卫生问题。