Cysteamine revisited: repair of arginine to cysteine mutations.

Cysteamine is a small aminothiol endogenously derived from coenzyme A degradation. For some decades, synthetic cysteamine has been employed for the treatment of cystinosis, and new uses of the drug continue to emerge. In this review, we discuss the role of cysteamine in cellular and extracellular homeostasis and focus on the potential use of aminothiols to reconstitute the function of proteins harboring arginine (Arg) to cysteine (Cys) mutations, via repair of the Cys residue into a moiety that introduces an amino group, as seen in basic amino acid residues Lys and Arg. Cysteamine has been utilized in vitro and ex vivo in four different genetic disorders, and thus provides "proof of principle" that aminothiols can modify Cys residues. Other aminothiols such as mercaptoethylguanidine (MEG) with closer structural resemblance to the guanidinium moiety of Arg are under examination for their predicted enhanced capacity to reconstitute loss of function. Although the use of aminothiols holds clinical potential, more studies are required to refine specificity and treatment design. The efficacy of aminothiols to target proteins may vary substantially depending on their specific extracellular and intracellular locations. Redox potential, pH, and specific aminothiol abundance in each physiological compartment are expected to influence the reactivity and turnover of cysteamine and analogous drugs. Upcoming research will require the use of suitable cell and animal models featuring Arg to Cys mutations. Since, in general, Arg to Cys changes comprise about 8% of missense mutations, repair of this specific mutation may provide promising avenues for many genetic diseases.

Folinic acid treatment for schizophrenia associated with folate receptor autoantibodies.

BACKGROUND: Auto-antibodies against folate receptor alpha (FRα) at the choroid plexus that block N(5)-methyltetrahydrofolate (MTHF) transfer to the brain were identified in catatonic schizophrenia. Acoustic hallucinations disappeared following folinic acid treatment. Folate transport to the CNS prevents homocysteine accumulation and delivers one-carbon units for methyl-transfer reactions and synthesis of purines. The guanosine derivative tetrahydrobiopterin acts as common co-factor for the enzymes producing dopamine, serotonin and nitric oxide. METHODS: Our study selected patients with schizophrenia unresponsive to conventional treatment. Serum from these patients with normal plasma homocysteine, folate and vitamin B12 was tested for FR autoantibodies of the blocking type on serial samples each week. Spinal fluid was analyzed for MTHF and the metabolites of pterins, dopamine and serotonin. The clinical response to folinic acid treatment was evaluated. RESULTS: Fifteen of 18 patients (83.3%) had positive serum FR auto-antibodies compared to only 1 in 30 controls (3.3%) (χ(2)=21.6; p<0.0001). FRα antibody titers in patients fluctuated over time varying between negative and high titers, modulating folate flux to the CNS, which explained low CSF folate values in 6 and normal values in 7 patients. The mean±SD for CSF MTHF was diminished compared to previously established controls (t-test: 3.90; p=0.0002). A positive linear correlation existed between CSF MTHF and biopterin levels. CSF dopamine and serotonin metabolites were low or in the lower normal range. Administration of folinic acid (0.3-1mg/kg/day) to 7 participating patients during at least six months resulted in clinical improvement. CONCLUSION: Assessment of FR auto-antibodies in serum is recommended for schizophrenic patients. Clinical negative or positive symptoms are speculated to be influenced by the level and evolution of FRα antibody titers which determine folate flux to the brain with up- or down-regulation of brain folate intermediates linked to metabolic processes affecting homocysteine levels, synthesis of tetrahydrobiopterin and neurotransmitters. Folinic acid intervention appears to stabilize the disease process.

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer.

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. The current dietary Phe restriction treatment has certain clinical limitations. To explore a long-term nondietary restriction treatment, a somatic gene transfer approach in a PKU mouse model (C57Bl/6-Pahenu2) was employed to examine its preclinical feasibility. 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. The blood Phe concentrations decreased to normal levels (< or =100 microM or 1.7 mg/dl) 2 weeks after vector application, independent of the sex of the PKU animals and the route of application. In particular, the therapeutic long-term correction in females was also dramatic, which had previously been shown to be difficult to achieve. Therapeutic ranges of Phe were accompanied by the phenotypic reversion from brown to black hair. In treated mice, PAH enzyme activity in whole liver extracts reversed to normal and neither hepatic toxicity nor immunogenicity was observed. 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. This study demonstrates the complete correction of hyperphenylalaninemia in both males and females with a rAAV serotype 8 vector. More importantly, the feasibility of a single intravenous injection may pave the way to develop a clinical gene therapy procedure for PKU patients.

Hyperphenylalaninemia and 7-pterin excretion associated with mutations in 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH: analysis of enzyme activity in intestinal biopsies.

Hyperphenylalaninemia, which can cause neurological disorders and mental retardation, results from a mutation in phenylalanine hydroxylase or an enzyme required for biosynthesis or regeneration of its cofactor, tetrahydrobiopterin. The hyperphenylalaninemia variant primapterinuria is characterized by the excretion of 7-biopterin (primapterin). This disorder is thought to be due to a deficiency of 4a-hydroxy-tetrahydrobiopterin dehydratase (pterin-4a-carbinolamine dehydratase), but a lack of tissue activity has not been directly demonstrated. The five mutations so far recognized in patients with primapterinuria are associated with either a single amino acid change or a premature stop codon. Only C81R has been successfully expressed in soluble form, and was found to have 40% of normal activity. Tissues which could be obtained by minimally invasive procedures were analyzed for dehydratase activity. None was detected in normal human white cells or fibroblasts. However, activity was found in intestine of rat, dog, pig, and particularly humans where it was only eight times lower than in liver. Distribution along the length and across the wall of small intestine was relatively uniform. Moreover, the dehydratases from human liver and intestinal mucosa have identical kinetic properties. A biopsy of duodenal mucosa from a patient with homozygous E96K dehydratase had activity of 55 nmol. min(-1)g(-1) mucosa compared to 329 +/- 32 nmol. min(-1)g(-1) tissue in controls (n = 12). The sixfold lower tissue activity of the E96K mutant alone may not be sufficient to account for the biochemical symptoms of primapterinuria in this patient. However, accumulation of a 4a-hydroxy-tetrahydrobiopterin degradation product (a side-chain cyclic adduct), which has been observed in vitro and appears to be a dehydratase inhibitor, may further exacerbate the problem.

Reconstitution of a metabolic pathway with triple-cistronic IRES-containing retroviral vectors for correction of tetrahydrobiopterin deficiency.

BACKGROUND: Tetrahydrobiopterin (BH4) is an essential cofactor for catecholamine and serotonin neurotransmitter biosynthesis. BH4 biosynthesis is carried out in a three-enzyme pathway involving GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Treatment of genetic defects leading to BH4 deficiency requires neurotransmitter replacement since synthetic cofactor does not efficiently penetrate the blood-brain barrier. Autologous fibroblasts transplanted into the brain as depository cells for drug delivery might offer an alternative. However, normal fibroblasts do not express GTPCH, and fibroblasts from PTPS patients lack two biosynthetic enzymes for BH4 production. METHODS: We engineered primary fibroblasts by the use of triple-cistronic, retroviral vectors for cofactor production. RESULTS: Constitutive SR activity in these cells enabled BH4 biosynthesis by transducing GTPCH and PTPS cDNAs together with a selective marker coupled in a single transcript with two IRES-elements in tandem. Upon reaching a critical concentration (> 400 pmol/mg protein) of intracellular BH4, the fibroblasts efficiently released cofactor even under non-dividing conditions. CONCLUSION: The use of triple-cistronic vectors for single transduction to reconstitute metabolic pathways or to treat multi-genetic diseases may be useful for engineering, for instance, depository cells for various organs, including the nervous system.

Overexpression of pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 in human colon cancer.

Pterin-4a-carbinolamine dehydratase (PCD) is a bifunctional protein also known as DCoH (dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)). PCD/DCoH modulates the DNA binding specificity of HNF1, thus acting on its transcriptional activity. In addition, it participates in the recycling of tetrahydrobiopterin (BH(4)), an essential cofactor of several metabolic reactions. We investigated colorectal tumors and colorectal tumor cell lines as compared to normal colon samples in search of a potential differential expression of PCD/DCoH. Immunohistochemistry was conducted on 20 human colorectal tumors and 20 normal samples using a specific polyclonal antibody. Immunoblotting and RT-PCR analysis for PCD/DCoH and HNF1 were also performed on both human tissues and CACO-2 and HT-29 cell lines. All of the 20 tumors and both colon cancer cell lines presented a strong and widespread immunoreactivity for PCD/DCoH, contrasting with the absence of expression in the normal epithelia. We thus report the massive overexpression of PCD/DCoH in colon tumors, which is in striking contrast with the absence of staining in normal counterparts. The sharp contrast in the expression of a modulator of transcriptional activity between tumoral and normal cells may have a physiopathological role. PCD/DCoH could potentially be a new marker of malignant colon cells in vivo.

Retrovirus-mediated double transduction of the GTPCH and PTPS genes allows 6-pyruvoyltetrahydropterin synthase-deficient human fibroblasts to synthesize and release tetrahydrobiopterin.

The tetrahydrobiopterin (BH4) cofactor is essential for the aromatic amino acid hydroxylases that are involved in phenylalanine degradation and catecholamine and serotonin biosynthesis. Furthermore, BH4 is an essential and limiting cofactor for all types of nitric oxide synthases. BH4 deficiency results in hyperphenylalaninemia and monoamine neurotransmitter depletion associated with progressive mental retardation and is most commonly due to autosomal recessive mutations in 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme for cofactor biosynthesis. Due to the relatively poor blood-brain barrier penetration of the cofactor, conventional therapy requires, besides oral doses of synthetic BH4, administration of neurotransmitter precursors and an aromatic amino acid decarboxylase inhibitor. The outcome of this therapy is not always beneficial. In this study we transduced into primary patient fibroblasts the human cDNAs for the BH4 biosynthetic enzymes GTP cyclohydrolase I and PTPS, expressed from different retroviral vectors. This allowed BH4 biosynthesis in originally PTPS-deficient cells. Moreover, the double-transduced fibroblasts released between 200 and 800 pmol of BH4/10(6) cells/day. Such engineered fibroblasts may be grafted into the central nervous system and used as depository cells for constitutive delivery of BH4.

Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families.

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.

Retrovirus-mediated gene transfer of 6-pyruvoyl-tetrahydropterin synthase corrects tetrahydrobiopterin deficiency in fibroblasts from hyperphenylalaninemic patients.

Tetrahydrobiopterin (BH4) deficiency, a variant form of hyperphenylalaninemia with progressive neurological dysfunction, is primarily caused by autosomal recessive mutations in the gene encoding the 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS is a biosynthetic enzyme for the BH4 co-factor, and its deficiency is associated with a malfunction of the phenylalanine catabolism in the liver and a lack of biogenic amine neurotransmitters dopamine and serotonin in the brain. We have previously isolated the wild-type PTPS cDNA and identified several mutations responsible for a decreased enzyme in patients. This study reports the in vitro correction of BH4 deficiency by using retrovirus mediated transfer of the PTPS cDNA into primary fibroblast cultures established from different patients. The Bing packaging cell line was used for amphotropic virus production. Following PTPS gene transfer, stimulation with cytokines restored biosynthesis of BH4 in originally defective cells to values comparable to those of heterozygous fibroblasts from clinically healthy subjects. These results not only provide a direct proof that the mutations in PTPS were causative for the mutant phenotype, but they are also the first step toward gene therapy as a potential alternative approach to treat BH4 deficiency.

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].

Characterization of the human PCBD gene encoding the bifunctional protein pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor for the transcription factor HNF-1 alpha.

The human pterin-4 alpha-carbinolamine dehydratase (PCD)/dimerization cofactor for the transcription factor HNF-1 alpha is a bifunctional protein proposed to be involved in entirely different biochemical functions. We previously established the complete amino acid sequence for the human liver PCD and subsequently isolated its corresponding cDNA. Using this cDNA as a probe, we isolated and determined the complete nucleotide sequence and flanking regions of the single human PCBD gene. The protein coding region of the gene is about 5 kb in length and contains 4 exons. We also defined the messenger RNA 5'-end by reverse transcription of the cap structure, thus allowing to analyze the promoter organization. Within the 5'-flanking sequence, potential regulatory regions include consensus binding sites for transcription factor Sp1, an AP-1, and several AP-2 binding sites; however, the 5' upstream region lacks both a proximal TATA and CAAT box promoter element.

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase.

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.

Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity.

6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.

The nifEN genes participating in FeMo cofactor biosynthesis and genes encoding dinitrogenase are part of the same operon in Bradyrhizobium species.

The nucleotide sequences of genes homologous to the Klebsiella pneumoniae nifEN genes have been determined in Bradyrhizobium japonicum 110. The coding regions for the nifE and nifN consist, respectively, of 1641 and 1407 nucleotides. The nifD gene (coding for the beta-subunit of dinitrogenase) and nifE are linked, and separated by 95 nucleotides. In the region of 12 nucleotides that separates nifE from nifN the stop codon for nifE overlaps the putative ribosome binding site for nifN. In contrast to Klebsiella and Azotobacter vinelandii, the B. japonicum nifEN genes are linked to the nifDK genes in the same operon. Comparison of dinitrogenase polypeptides (nifDK products) and the polypeptides of the nifE and nifN genes reveals considerable homology between nifD and nifE, and between nifK and nifN. Several protein domains, containing highly conserved cysteine residues, are conserved among the gene products of nifD, nifK, nifE and nifN. This result allows us to propose a probable evolutionary pathway for the common origin of these genes.