The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones.