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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：
9981023
负责人：
EILEEN K JAFFE
金额：
$ 39.79万
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 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9981023
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 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 in silico 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.

项目总结