Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7-8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.
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PMID:Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system. 892 Oct 3

Upon birth, the liver acquires new functions as a result of the initiation of expression of key enzymes. One example is the initiation of gluconeogenesis which depends on the induced appearance of phosphoenolpyruvate carboxykinase (P-pyruvate-CK) at birth. To characterize other genes that undergo such regulation, a differential screening was performed on a cDNA library from well-differentiated hepatoma cells. The pattern of tissue-specific and developmental-specific expression was determined for seven genes. Three clones, out of which two encode for the known genes alcohol dehydrogenase class I (ADH) and phenylalanine 4-monooxygenase (PAH) and a new gene (clone 116-3), exhibited a pattern of expression similar to that of the P-pyruvate-CK gene, i.e. their expression was liver and kidney specific and induced in the liver upon birth. Determination of the sequence of clone 116-3 revealed that it belonged to the UDP-glucuronosyltransferases type 2 (UGT2) family and thus was named UGT2B-rH4. To examine whether expression of the various genes could be prematurely induced by hormones in the fetal liver, either high levels of cAMP or low levels of insulin were induced in utero. The results demonstrated that cAMP induced a marked expression only of the genes for P-pyruvate-CK and ADH but not of those for PAH or UGT2B-rH4, while insulin deficiency induced premature expression of all four genes. We suggest that a set of genes whose expression is specifically induced in the liver upon birth can be prematurely induced by the hormones in utero.
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PMID:Identification of differentially expressed genes during hepatocytes development and characterization of their prenatal hormonal induction. 902 81

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.
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PMID:Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families. 922 57

An A-->T substitution in cDNA nucleotide 1197 (c.1197A/T) of the human phenylalanine hydroxylase (PAH) gene has been regarded as a silent mutation, because both the wild-type (GUA) and the mutant (GUU) alleles encode a valine residue at codon 399 (V399 V). The nucleotide c.1197 is located at the 3'-end of exon 11at position -3 of the exon-intron junction. To explore whether the substitution exerts any effects on the processing of the PAH mRNA, illegitimate PAH transcripts from lymphoblast cultures of a phenylketonuria (PKU) patient heterozygous for c.1197A/T were analyzed by the polymerase chain reaction following reverse-transcription (RT-PCR). mRNAs with an exon 11 deletion were revealed. Furthermore, by using an R408 W mutation in the paternal allele as a marker, sequence analysis of the RT-PCR products indicates that virtually all PAH transcripts from the maternal allele with the c. 1197A/T substitution do not contain exon 11. To address whether this substitution is the main determinant for exon skipping, PAH minigenes with or without the substitution were constructed and transfected to a human hepatoma cell line. Analysis of the transcription products by S1 nuclease mapping clearly indicated that such exon 11 skipping was directly associated with the c.1197A/T substitution. Thus, this study demonstrates that the c.1197A/T substitution in the PAH gene is not just a neutral polymorphism but a mutation that induces post-transcriptional skipping of exon 11 leading to a PKU phenotype.
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PMID:A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria. 1121 2

We have uncovered a fundamental difference in the regulation of the rodent and the human phenylalanine hydroxylase (PAH) genes: expression of human PAH is independent of glucocorticoids and/or cAMP in contrast to the mouse gene which is not only highly inducible but dependent upon hormones for expression. Nevertheless, the two genes do exhibit similarities: DNaseI hypersensitive sites are identically located in the regulatory regions, and the sequences around these sites are partially conserved and associated with regulatory elements sharing similar function. In transient transfections, the human proximal promoter is tissue-specific and presents significant activity compared to the extremely low and ubiquitous activity of the mouse promoter. DNA fragments corresponding to the two upstream hypersensitive sites of both genes have enhancer activity that depends upon the liver-enriched transcription factor binding sites for hepatocyte nuclear factor (HNF) 1 and/or CCAAT/enhancer binding protein (C/EBP). While expression of the rodent gene relies upon two modules in the HSIII enhancer, one activated by HNF1 and C/EBP and the other required for the hormone response, the human equivalent has conserved only the liver-specific transcription factor binding module. Even though the more proximal enhancer is not necessary for full reporter gene activity in transient transfection assays in Pah-expressing hepatoma cells, this enhancer could be required in both species for activation during development.
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PMID:Conserved as well as divergent regulatory elements account for expression of the human and rodent phenylalanine hydroxylase genes. 1167 21

Tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency is a recently recognized variant of phenylketonuria, with a probable multifactorial molecular basis. In this study we have investigated the effect of BH4 on PAH gene expression in human hepatoma. Our results show that increased BH4 levels result in an enhancement of PAH activity and PAH protein, due to longer turnover rates, while PAH mRNA levels remain unchanged. This was confirmed for mutant PAH proteins (A309V, V388M and Y414C) associated to in vivo BH4 responsiveness, validating previous studies. We can conclude that there is no effect of the cofactor on PAH gene transcription, probably being the chemical chaperone effect of BH4 stabilizing mutant PAH proteins the major underlying mechanism of the response.
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PMID:Analysis of the effect of tetrahydrobiopterin on PAH gene expression in hepatoma cells. 1650 82

Phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, is caused by mutations in human phenylalanine hydroxylase gene (PAH), leading to deficient enzyme activity. Previously reported but uncharacterized PAH gene mutation, p.S231F (c.692C > T), was detected in Serbian patients with classical PKU. We analyzed p.S231F PAH protein in prokaryotic (Escherichia coli) and eukaryotic expression system (hepatoma cells). In both systems the mutant enzyme was unstable. Residual enzyme activity in vitro was approximately 1%. Mutation p.S231F PAH was not activated by pre-incubation with phenylalanine substrate. We found no GroEL/GroES chaperone effect and slightly positive effect of the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) on the stabilization of the protein structure. Our findings were in accordance with severe patients' phenotypes. In conclusion, p.S231F should be classified as a functionally null PAH gene mutation as it drastically reduces stability and activity of the PAH enzyme in vitro.
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PMID:The Missense p.S231F phenylalanine hydroxylase gene mutation causes complete loss of enzymatic activity in vitro. 1962 56

We have previously demonstrated the efficacy of antisense therapy for splicing defects in cellular models of metabolic diseases, suppressing the use of cryptic splice sites or pseudoexon insertions. To date, no animal models with these defects are available. Here, we propose exon skipping of the phenylalanine hydroxylase (Pah) gene expressed in liver and kidney to generate systemic hyperphenylalaninemia in mice as a sensitive in vivo assay to test splice suppression. Systemic elevation of blood L-Phe can be quantified using tandem MS/MS. Exon 11 and/or 12 skipping for the normal PAH gene was validated in hepatoma cells for comparing two oligonucleotide chemistries, morpholinos and locked nucleic acids. Subsequently, Vivo-morpholinos (VMO) were tested in wild-type and in phenotypically normal Pah(enu2/+) heterozygous mice to target exon 11 and/or 12 of the murine Pah gene using different VMO dosing, mode of injection and treatment regimes. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. Sustained effect required repeated injection of VMOs. Our results provide not only a sensitive in vivo assay to test for splice-modulating antisense oligonucleotides, but also a simple method to generate murine models for genetic liver diseases.
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PMID:A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver. 2522 62

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)-one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.
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PMID:Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model. 2965 78

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.
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PMID:E3 Ubiquitin Ligase APC/CCdh1 Regulation of Phenylalanine Hydroxylase Stability and Function. 3326 Jun 74


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