Disease relevance of Pah

• A recombinant adeno-associated virus (rAAV) vector containing the murine Pah-cDNA was generated, pseudotyped with capsids from AAV serotype 8, and delivered into the liver of PKU mice via single intraportal or tail vein injections [1].
• Phenylketonuria (PKU) is an inborn error of metabolism caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which leads to high blood phenylalanine (Phe) levels and consequent damage of the developing brain with severe mental retardation if left untreated in early infancy [1].
• In treated mice, PAH enzyme activity in whole liver extracts reversed to normal and neither hepatic toxicity nor immunogenicity was observed [1].
• We have demonstrated the cognitive outcome of biochemical and phenotypic reversal by the adeno-associated virus vector-mediated gene delivery of a human PAH transgene [2].
• Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly [3].

High impact information on Pah

• Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA [3].
• Identification of hepatic nuclear factor 1 binding sites in the 5' flanking region of the human phenylalanine hydroxylase gene: implication of a dual function of phenylalanine hydroxylase stimulator in the phenylalanine hydroxylation system [4].
• To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity [5].
• Genetic mapping has localized the mutation to murine chromosome 10 at or near the Pah locus, the structural gene for phenylalanine hydroxylase [6].
• We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal [6].

Chemical compound and disease context of Pah

• Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine [7].
• BH4-responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine [8].
• Continuously growing populations of cells resembling the parental MEL cells and expressing mouse phenylalanine hydroxylase were obtained only when rat hepatoma cells treated with mitomycin or iodoacetamide, which inactivate DNA and SH proteins, respectively, were fused with MEL cells [9].
• BWTG3 hepatoma cells can acquire phenylalanine hydroxylase, cystathionine synthase and CPS-I without genetic manipulation, but activation of the silent OTC gene requires cell fusion with hepatocytes [10].
• P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria [11].

Biological context of Pah

• Various mutant hyperphenylalaninemic mouse models with an HPA phenotype, generated by N-ethyl-N'-nitrosourea (ENU) mutagenesis at the Pah locus, have become available [12].
• With the long-term goal of using gene transfer to the skin to remove phenylalanine, we have previously shown that overexpression of PAH, catalyzing the hydroxylation of phenylalanine, and GTP cyclohydrolase (GTP-CH), involved in the formation of the necessary cofactor BH4,are required [13].
• We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein [7].
• The rate of hepatic PAH enzyme activity increased significantly with BH4 content without affecting gene expression or Pah-mRNA stability [8].
• The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver [14].

Anatomical context of Pah

• Phenylalanine hydroxylase (PAH) activity was constitutively expressed in skeletal and cardiac muscle of transgenic mice which carried the PAH cDNA under the transcriptional control of the mouse muscle creatine kinase promoter [15].
• Phenylketonuria (PKU) is an inherited disease causing increased levels of phenylalanine in body fluids due to deficiency of hepatic phenylalanine hydroxylase (PAH) or other enzymes involved in the phenylalanine metabolism [13].
• The PAH enzyme activity was titrated by employing mutant PAH enzymes with different specific activity and by increasing the PAH copy number in transduced keratinocytes using a repeated transduction procedure [13].
• PAH activity measured in intact marrow cells was significantly lower than in marrow lysates; no such difference was measured in isolated hepatocytes vs. liver homogenate [16].
• Our specific hypothesis was that phenylalanine hydroxylase (PAH) expressed in bone marrow would lower blood phenylalanine levels in hyperphenylalaninemic Pah(enu2) mice, a model of human phenylketonuria (PKU) [16].

Associations of Pah with chemical compounds

• In contrast, a lentiviral vector expressing the murine Pah-cDNA, delivered via intraportal vein injection into PKU mice, did not result in therapeutic levels of blood Phe [1].
• The Pah enzyme activities of the various models correlate inversely with the corresponding phenylalanine levels in plasma and brain and the delay in plasma clearance response following a phenylalanine challenge [12].
• These mice exhibited hyperphenylalaninemia at baseline, but serum phenylalanine levels decreased significantly when the mice were supplemented with tetrahydrobiopterin (BH4), a required cofactor for PAH [15].
• Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH4 on an additional level and provide a molecular explanation for cofactor-responsive PKU [8].
• The loading regimen was also used to gauge the response of mutant hyperphenylalaninemic mice to exposure to chemical compounds required for normal phenylalanine catabolism (i.e. pteridine cofactors of the phenylalanine hydroxylase reaction) [17].

Regulatory relationships of Pah

• We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice [18].
• Tryptophan hydroxylase from mast cells was considerably inhibited by o-phenanthroline like phenylalanine hydroxylase as well as tyrosine hydroxylase from other sources, and its Ki was between 1.2 muM and 4.53 muM [19].

Other interactions of Pah

• Characterization of transgenic mice with the expression of phenylalanine hydroxylase and GTP cyclohydrolase I in the skin [20].
• However, low level of phenylalanine clearance was observed in mice expressing PAH and GTP-CH from the K14 promoter, suggesting that the skin can be genetically engineered to function as a 'metabolic sink' [20].
• Mouse erythroleukemia (MEL) cells do not synthesize any detectable level of phenylalanine hydroxylase and thus do not grow in Tyr- medium [9].
• There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase [21].
• Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes [22].

Analytical, diagnostic and therapeutic context of Pah

• The efficacy of bone-marrow-directed gene therapy as a metabolic sink in the treatment of phenylketonuria may be limited, although further experiments with greater marrow PAH expression levels will be necessary to definitively prove this conclusion [16].
• Phenylalanine clearance may have been limited by the natural perfusion rate of the marrow compartment, by insufficient PAH expression in marrow, or by other cellular factors affecting phenylalanine metabolism in intact marrow cells [16].
• Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein [21].
• In the present study, we developed a breath test for mice using [1-13C]phenylalanine in order to examine the BH4 responsiveness of normal PAH in vivo [23].
• The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents [24].

References