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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要苯丙氨酸羟化酶（PAH）功能障碍是氨基酸代谢中最常见的先天性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 Phe 维持在足以进行蛋白质生物合成的水平，但低于神经毒性水平。规定是通过 Phe 的变构激活来完成。根据对 PKU 患者的广泛研究，目前的医学共识是终生控制血液 Phe 水平以达到并维持正常神经功能；这表明需要更好地理解 PAH 结构/功能关系以支持对现有 PAH 药理学伴侣的了解以及新型药物的未来发展非饮食疗法。 2013年我们推出了创新的PAH变构构象选择模型其中包括一个静息态四聚体、一个结构独特的激活四聚体和更小的组装体；只有活化的 PAH 才含有变构 Phe 结合位点。该位点位于多聚体特异性亚基-亚基处接口，其细节仍未知。我们的模型包括以前未预见到的域旋转，最近发表的生物物理学研究有力地支持了这一点。 2016 年我们出版了全长静息态哺乳动物 PAH 的第一个晶体结构；这是对该领域期待已久的贡献。小角度 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。当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。我们测试是否与特定的常见疾病相关的 PAH 变异体在静息态和激活态 PAH 之间的转换方面存在缺陷，因此对 Phe 的变构激活。这一假设与北京大学作为一所大学的传统观点大相径庭。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大鼠 PAH 的工作扩展到人类 PAH。在 AIM 3 我们确定可以通过稳定以下物质来调节 PAH 功能（消极或积极）的物质：静息状态或激活的 PAH。使用体外方法，我们将筛选批准的药物和环境污染物，接触这些污染物可能会混淆 PKU 表型。我们使用计算机筛选文库类药物分子为未来开发新的 PKU 疗法提供线索。所有 AIMS 均雇用建立了评估野生型、疾病相关型和设计型 PAH 的生化和生物物理方法从静止状态过渡到激活状态的变体。关键方法包括内在蛋白质荧光、 SAXS、分析超速离心、晶体学、非变性 PAGE、酶动力学以及创新使用离子交换色谱法可解析构象不同的 PAH 多聚体。我们的广泛方法将产生新的重要信息，可用于更好地了解 PKU 的分子基础。