A New View of PAH Allostery - Correlation with Disease-Associated Alleles

PAH 变构的新观点 - 与疾病相关等位基因的相关性

• 批准号: 9547552
• 负责人: EILEEN K JAFFE
• 金额: $ 39.12万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9547552
• 关键词: Achievement; Active Sites; Address; Affect; Alleles; Allosteric Regulation; Allosteric Site; Architecture; Basic Science; Binding; Binding Sites; Biochemical; Birth; Blood; Cell Culture System; Classical phenylketonuria; Computer Simulation; Consensus; Crystallization; Crystallography; Development; Disease; Environmental Pollution; Enzyme Kinetics; Enzymes; Equilibrium; Exposure to; Fluorescence; Functional disorder; Future; Genotype; Heterogeneity; Heterozygote; Homology Modeling; Human; Hyperphenylalaninaemias; In Vitro; Inborn Errors Amino Acid Metabolism; Inborn Errors of Metabolism; Individual; Ion-Exchange Chromatography Procedure; Knowledge; Length; Libraries; Life; Medical; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Nervous System Physiology; Neurologic Dysfunctions; Online Mendelian Inheritance In Man; Pharmaceutical Preparations; Pharmacology; Phenotype; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Physiological; Population; Porphobilinogen Synthase; Protein Biosynthesis; Proteins; Publications; Publishing; Rattus; Regulation; Research; Rest; Roentgen Rays; Rotation; Shapes; Site; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic; Tyrosine; Variant; Work; X-Ray Crystallography; analytical ultracentrifugation; base; biophysical analysis; biophysical techniques; design; dimer; improved; innovation; molecular shape; mouse model; neurobehavioral; neurotoxic; novel; novel therapeutics; predictive modeling; prevent; protein folding; protein intake; reproductive; response; screening; small molecule; social; therapeutic development

PROJECT SUMMARY Dysfunction of phenylalanine hydroxylase (PAH) is the most common inborn error of amino acid metabolism and the underlying cause of phenylketonuria (PKU). By converting phenylalanine (Phe) to tyrosine, PAH maintains blood Phe at levels sufficient for protein biosynthesis, but below neurotoxic levels. Regulation is accomplished by allosteric activation by Phe. Based on extensive studies of individuals living with PKU, the current medical consensus is to control blood Phe levels throughout life to achieve and maintain normal neurological function; this argues for a better understanding of PAH structure/function relationships to support both the understanding of existing pharmacological chaperones for PAH and the future development of novel non-dietary therapeutics. In 2013 we introduced an innovative conformational selection model of PAH allostery that includes a resting-state tetramer, an architecturally distinct activated tetramer, and smaller assemblies; only activated PAH contains the allosteric Phe binding site. This site is at a multimer-specific subunit-subunit interface, the details of which remain unknown. Our model includes a previously unforeseen domain rotation, which is now strongly supported by recently published biophysical studies. 2016 marks our publication of the first crystal structure for full length resting-state mammalian PAH; this is a long-awaited contribution to the field. Small angle X-ray scattering (SAXS) supports both resting state PAH and Phe-stabilized activated PAH tetramer structures, and confirms a major conformational difference between the two, which is consistent with our allosteric model. The current application builds on these achievements. In AIM 1 we address the relevance of our allosteric model to disease. We test whether specific common disease-associated PAH variants are defective in the transition between resting-state and activated PAH and thus insensitive to allosteric activation by Phe. This hypothesis is a major departure from the conventional view of PKU as a protein folding/stability disorder. In AIM 2 we determine the structure of activated PAH using X-ray crystallography and SAXS, and we extend our work with rat PAH to human PAH using a designed variant. In AIM 3 we identify substances that can modulate PAH function (negatively or positively) by stabilizing either resting-state or activated PAH. Using in vitro methods, we will screen approved drugs and environmental contaminants, exposure to which can confound PKU phenotype. We use in silico screening of libraries of drug-like molecules to provide leads for future development of new PKU therapies. All AIMS employ established biochemical and biophysical methods to assess wild-type, disease-associated, and designed PAH variants for the transition from resting to activated states. Key methods include intrinsic protein fluorescence, SAXS, analytical ultracentrifugation, crystallography, native PAGE, enzyme kinetics, and the innovative use of ion exchange chromatography to resolve conformationally distinct PAH multimers. Our broad approach will yield new and important information applicable to a better understanding of the molecular bases for PKU.

项目总结 苯丙氨酸羟化酶(PAH)功能障碍是氨基酸代谢中最常见的先天错误 以及苯丙酮尿症(PKU)的根本原因。通过将苯丙氨酸(Phe)转化为酪氨酸，PAH 将血液中的苯丙氨酸维持在蛋白质生物合成所需的水平，但低于神经毒性水平。监管是 通过苯丙氨酸的变构活化完成的。基于对北大患者的广泛研究， 目前的医学共识是在一生中控制血液中的Phe水平，以达到并保持正常 神经功能；这为更好地理解PAH结构/功能关系提供了支持 对多环芳烃现有药理伴侣的认识及未来的发展 非饮食疗法。2013年，我们引入了一种创新的多环芳烃变构构象选择模型 这包括休眠状态的四聚体、结构上不同的活化四聚体和较小的组装体； 只有活化的PAH才含有变构Phe结合部位。该站点位于特定于多聚体的亚基-亚基 接口，具体细节尚不清楚。我们的模型包括以前未预见到的区域旋转， 最近发表的生物物理研究有力地支持了这一观点。2016年是我们出版的 第一个全长静息态哺乳动物多环芳烃的晶体结构；这是该领域期待已久的贡献。 小角X射线散射(SAXS)支持静止态PAH和Phe稳定的活化PAH 四聚体结构，并证实了两者之间的主要构象差异，这与 我们的变构模型。目前的应用程序建立在这些成就的基础上。在目标1中，我们解决了 我们的变构模型与疾病的相关性。我们测试了与特定常见疾病相关的PAH 变异体在静息状态和激活的多环芳烃之间的转换过程中存在缺陷，因此对 苯丙氨酸的变构活化。这一假设与传统的将北大视为 蛋白质折叠/稳定性障碍。在AIM 2中，我们用X射线确定了活化的多环芳烃的结构 结晶学和SAXS，我们使用设计的变体将我们对大鼠PAH的工作扩展到人PAH。在……里面 目的3我们确定可以通过稳定以下两种方式调节多环芳烃功能的物质： 静息状态或激活的多环芳烃。使用体外方法，我们将筛选批准的药物和环境 污染物，暴露在这些污染物中会混淆PKU的表型。我们在电子筛选库中使用了 类药物分子，为未来开发新的PKU疗法提供线索。所有AIMS员工 建立生化和生物物理方法来评估与疾病相关的野生型和设计的多环芳烃 从静息状态转换到激活状态的变体。关键方法包括本征蛋白质荧光， SAXS、分析超速离心法、结晶学、天然PAGE、酶动力学，以及 离子交换层析用于拆分构象不同的多环芳烃多聚体。我们广泛的方法将 产生适用于更好地理解PKU的分子基础的新的和重要的信息。