RESUMEN
Phenylketonuria (PKU) is an autosomal recessive disorder caused by a defective phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr) in presence of the cofactor tetrahydrobiopterin (BH4). Defective PAH causes accumulation of phenylalanine, which has neurotoxic effects and leads to dermatological, behavioral, and neurocognitive problems. Treatments for this disease consist in life-long diets that are hard for patients to keep, or supplementation with BH4. In this study, we propose a system where a probiotic lactic acid bacteria (LAB) can be used as vehicle to express in situ an engineered human PAH. Engineered PAHs contain a secretion peptide, a gastrointestinal signal (GI), the human PAH, and a flexible glycine linker followed by the fluorescence protein mEGFP. Engineered constructs were successfully transformed, expressed, and secreted in Lactobacillus plantarum CM_PUJ411. PAH construct containing either the signal peptide GI1 or GI2 were transported through a Caco-2 cell monolayer. Nevertheless, the one containing GI1 allowed the highest transport through the cell monolayer. Co-culture of L. plantarum and Caco-2 cells showed that engineered PAH is produced in-situ and transported through the cell monolayer. Finally, the activity test showed that the engineered PAH secreted by L. plantarum CM_PUJ411 is active, leading to a reduction in l-Phe and an increase in l-Tyr levels, respectively. These results show the potential of this system as a new therapeutic alternative for the treatment of PKU patients.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lactobacillus plantarum/metabolismo , Fenilalanina Hidroxilasa/biosíntesis , Probióticos/administración & dosificación , Células CACO-2 , Tracto Gastrointestinal/metabolismo , Humanos , Lactobacillus plantarum/genética , Fenilalanina Hidroxilasa/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The effect of dietary protein concentrations on the hepatic expression of phenylalanine hydroxylase (PAH) and tyrosine aminotransferase (TAT) mRNA concentrations was studied in rats adapted to consume diets (18 or 50% casein) in a restricted schedule of 7 h (0900 to 1600) for 5 days. After 6 hours of feeding, TAT mRNA concentrations of rats adapted to 18% casein diet and fed acutely 6, 18 and 50% casein diet were 0.15, 0.84 and 5.08 fold respectively higher than mRNA concentrations of rats before feeding. After 17 hours of fasting, TAT mRNA concentrations of rats previously fed 6, 18 or 50% casein diet were -0.45, 1.76 and 9.11 fold respectively higher than mRNA concentrations of rats before they were fed. PAH mRNA concentrations showed a similar pattern. There was a -0.68, 1.63 and 2.5 fold rise of PAH mRNA concentrations in rats fed 6,18 and 50% casein diet during the feeding period, and -0.86, 2.32 and 9.33 fold rise after 17 hours of fasting. TAT and PAH mRNA concentrations of rats adapted to consume 50% casein diet and then changed to 6% or kept on the 50% casein diet showed a maximum peak 6 hours after the rats began to consume the diet; however, they decreased 5 hours after fasting. These results suggest that increasing concentrations of protein in the diet were able to increase the concentration of TAT and PAH mRNA, possibly in order to eliminate the excess of amino acids consumed. The concentration of TAT and PAH mRNA depended more on the protein content of the diet to which the rats were previously adapted.