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: 9350419
• 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/9350419
• 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; Dimerization; 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.

项目摘要 苯丙氨酸羟化酶（PAH）功能障碍是最常见的先天性氨基酸代谢缺陷 和苯丙酮尿症（PKU）的根本原因。通过将苯丙氨酸（Phe）转化为酪氨酸，PAH 维持血液Phe在足以进行蛋白质生物合成的水平，但低于神经毒性水平。监管是 通过Phe的变构激活完成。基于对PKU患者的广泛研究， 目前的医学共识是在整个生命过程中控制血液Phe水平，以实现并维持正常的Phe水平。 神经功能;这表明需要更好地理解PAH结构/功能关系，以支持 对PAH现有药理学伴侣的理解和新药物的未来发展， 非饮食疗法。2013年，我们引入了一种创新的PAH变构构象选择模型 包括静止状态的四聚体、结构上不同的活化四聚体和更小的组装体; 只有活化的PAH含有变构Phe结合位点。该位点位于多聚体特异性亚基-亚基 接口，其细节仍然未知。我们的模型包括一个以前无法预见的域旋转， 最近发表的生物物理研究强烈支持这一观点。2016年标志着我们的出版物 第一个晶体结构的全长静息态哺乳动物PAH;这是一个期待已久的贡献领域。 小角X射线散射（SAXS）支持静息态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AIM 1中，我们解决了 我们的变构模型与疾病的相关性。我们检测特定常见疾病相关PAH是否 变体在静息状态和活化PAH之间的转换中有缺陷，因此对 Phe的变构激活。这一假设是一个重大的背离传统的看法，北京大学作为一个 蛋白质折叠/稳定性障碍。在AIM 2中，我们使用X射线确定活化PAH的结构 晶体学和SAXS，我们使用设计的变体将我们的工作从大鼠PAH扩展到人类PAH。在 目的3：我们确定了可以调节PAH功能（负或正）的物质， 静息状态或激活PAH。使用体外方法，我们将筛选批准的药物和环境 污染物，暴露于其可混淆PKU表型。我们使用计算机筛选文库， 药物样分子，为未来开发新的PKU疗法提供线索。所有AIMS员工 建立了评估野生型、疾病相关和设计PAH的生化和生物物理方法 用于从静止状态到激活状态的转变的变体。关键方法包括内在蛋白质荧光， SAXS、分析超离心、晶体学、非变性PAGE、酶动力学以及 通过离子交换色谱法分离构象不同的PAH多聚体。我们的广泛方法将 产生新的和重要的信息，适用于更好地了解PKU的分子基础。