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.

项目摘要苯丙氨酸羟化酶（PAH）的功能障碍是氨基酸代谢最常见的人类误差以及苯酮尿症（PKU）的根本原因。通过将苯丙氨酸（PHE）转换为酪氨酸，PAH 保持血液的水平足以使蛋白质生物合成，但低于神经毒性水平。调节是通过PHE的变构激活完成。基于对PKU患者的广泛研究，当前的医疗共识是控制血液中的血液水平，以达到和维持正常神经功能；这说明对PAH结构/功能关系有更好的理解以支持对现有的药理学伴侣的理解以及新颖的未来发展非乳腺治疗学。 2013年，我们引入了PAH变构的创新构象选择模型其中包括一个静止状态四聚体，一个建筑截然不同的激活的四聚体和较小的组件；仅活化的PAH包含变构PHE结合位点。该站点位于多聚体特异性亚基套件接口，其细节仍然未知。我们的模型包括以前无法预见的域旋转，现在，最近发表的生物物理研究得到了强有力的支持。 2016年标志着我们的出版全长休息状态哺乳动物PAH的第一晶体结构；这是对该领域的期待已久的贡献。小角度X射线散射（SAX）支持静止状态PAH和PHE稳定的激活PAH 四聚体结构，并确认两者之间的主要构象差异，这与我们的变构模型。当前的应用程序基于这些成就。在目标1中，我们解决了我们的变构模型与疾病的相关性。我们测试特定的常见疾病相关的PAH是否变体在静止状态和活化的PAH之间的过渡中有缺陷，因此对 PHE的变构激活。这个假设与PKU的常规观点是一个主要的不同蛋白质折叠/稳定性障碍。在AIM 2中，我们使用X射线确定激活PAH的结构晶体学和SAXS，我们使用设计的变体将其与Rat Pah的工作扩展到人类PAH。在 AIM 3我们通过稳定两者来确定可以（负或积极地）调节PAH功能的物质静止状态或激活的PAH。使用体外方法，我们将筛选批准的药物和环境污染物，可能混淆PKU表型的暴露。我们用于筛选库的库类似药物的分子为未来开发新的PKU疗法提供潜在客户。所有目标都雇用已建立的生化和生物物理方法评估野生型，与疾病相关和设计的PAH 从静止状态到激活状态的过渡的变体。关键方法包括内在蛋白质荧光， SAX，分析性超速离心，晶体学，本地页面，酶动力学以及创新的使用离子交换色谱法解决构象上不同的PAH多聚体。我们广泛的方法将产生适用于PKU分子碱基的新的重要信息。