Function and regulation of phenylalanine and tyrosine hydroxylases from human and Caenorhabditis elegans. With focus in evolutionary aspects and development of new therapies

The family of the aromatic amino acid hydroxylases (AAAH) is well studied in mammals. It includes four members, i.e. phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPH1 and 2). These enzymes share important features, such as domain organization, three dimensional structure and mechanism of the reaction. The AAAH have important functions and are related to genetic human diseases. PAH, expressed in liver, is in charge of L-Phe catabolism from the diet and mutations in the PAH gene lead to phenylketonuria (PKU), a paradigm for genetic metabolic diseases. TH and the TPHs are enzymes of the neuroendocrine system, that carry out the rate limiting steps in the synthesis of neurotransmitters and hormones, i.e. catecholamines (TH) and serotonin and melatonin (TPHs). Mutations in TH and the TPHs genes are also involved in important neurological diseases and disorders, such as some forms of dystonia and parkinsonism in the case of TH and mood disorders in TPH. The nematode Caenorhabditis elegans is a model organism widely used in biology. We here present the expression and characterization of two AAAH from the nematode, PAH and TH, in order to get insights into evolution of structure, function and regulation in this enzymatic family. In the case of PAH we found functional and molecular similarities between C. elegans PAH (cePAH) and human PAH (hPAH), although they display important differences in enzymatic activity regulation, especially regarding the regulation exerted by the substrate, L-Phe. Both the preactivation and the positive cooperativity induced by the substrate on mammalian PAHs were absent in cePAH. In vivo experiments with knock-out worms bearing a deletion in the pah gene (pah-1) demonstrated that cePAH is involved in the synthesis of a melanin-like compound that localizes in the cuticle. The study of the recombinant TH from C. elegans (ceTH) in comparison with human TH (hTH) also revealed important differences at the level of short-term activity regulation. Basic regulatory mechanisms for hTH, such as substrate inhibition and feed-back inhibition by the end catecholamine products, appear to be absent in ceTH, suggesting a less tight regulation of enzymatic activity in the worm. But interestingly, ceTH was effectively phosphorylated by cAMPdependent protein kinase (PKA) at Ser35, though this modification did not translate into activation of the enzyme in synergy with the feed-back inhibition by catecholamines, as it is the case for phosphorylation of hTH at the equivalent Ser40. We hypothesised that phosphorylation of ceTH could regulate the interaction with other proteins and/or control subcellular localization. Supplementation with BH4 has been recently established as a therapeutic intervention for PKU. A main effect of the cofactor is the stabilisation of misfolded PKU mutants, and BH4 appears to function as a natural chaperone molecule. Since BH4 is a shared cofactor by all the AAAH we set to investigate the effect of BH4 supplementation on rat brain TH. Higher doses of BH4 than those currently used for the treatment of PKU were needed to increase the cofactor concentration in brain, most probably due to the selectivity of the blood-brain-barrier (BBB). This indicates that the current treatments using lower doses of BH4 (up to 20 mg/kg/day) are not expected to affect neuronal TH and TPH2. An increment of total TH protein and activity was measured in the brains of wild-type (wt) mice upon treatment with 100 mg BH4/kg/day, suggesting that BH4 also functions as a natural chaperone in the case of TH. In agreement with these effects, in vitro experiments also showed the capability of BH4 to stabilise TH. Finally, the screening of chemical libraries of small organic compounds is arising as a promising tool to find specific stabilisers of proteins (i.e. pharmacological chaperones). In the case of PAH, four stabilising compounds (compounds I-IV) were found in a previous study, revealing their potential as therapeutic pharmacological chaperones for PKU. As in the case of BH4, it was interesting to study the effect of these four molecules upon neuronal TH and TPH2. We found that compound III stabilized the three AAAH investigated, whereas the other compounds exerted different enzyme specific effects. In vivo studies with supplemented mice revealed the potential of compound III to treat TH-associated diseases. These results are important not only for the development of new specific therapies, but also to unravel enzyme specific/non specific ligand binding in the AAAH family