Wen Ni Chang

Grape seed extract inhibits the growth and pathogenicity of Staphylococcus aureus by interfering with dihydrofolate reductase activity and folate-mediated one-carbon metabolism

Staphylococcus aureus (S. aureus) is one of the most common pathogens that causes infectious and foodborne diseases worldwide. Searching for drug and chemical compounds against this bacterium is still in demand. We found that grape seed extract (GSE), a natural food product rich in polyphenols, inhibited the dihydrofolate reductase activity and growth of S. aureus. In addition, the intracellular content of tetrahydrofolate (THF), the major folate species identified in S. aureus, was significantly decreased when GSE was present in medium. The GSE-induced growth inhibition was reversed by adding, THF, 5,10-methylenetetrahydrofolate or methionine to the medium. The differential rescuing effects elicited by thymidine and methionine indicated that GSE-induced perturbation in folate-mediated one-carbon metabolism has more profound impact on methionine cycle than on thymidine monophosphate (TMP) synthesis. Significantly reduced inflammatory responses and mortality were observed in zebrafish infected with S. aureus pre-incubated with GSE. We conclude that GSE might serve as an effective natural alternative for the control of food poisoning caused by S. aureus with proper safety measure.

Characterization and Comparative Studies of Zebrafish and Human Recombinant Dihydrofolate Reductases—Inhibition by Folic Acid and Polyphenols

Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

Serine hydroxymethyltransferase (SHMT) provides activated one-carbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N5,N10-methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N5-CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.

Zebrafish larvae exposed to ginkgotoxin exhibit seizure-like behavior that is relieved by pyridoxal-5′-phosphate, GABA and anti-epileptic drugs

SUMMARY The etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5′-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.

Recombinant zebrafish γ-glutamyl hydrolase exhibits properties and catalytic activities comparable with those of mammalian enzyme

A cDNA encoding for zebrafish γ-glutamyl hydrolase (γGH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-zγGH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant zγGH was similar to mammalian γGH. Thrombin digestion of this NH-zγGH fusion protein resulted in zγGH with approximately 2-fold higher catalytic activity compared with the NH-zγGH fusion enzyme. This recombinant zγGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zγGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared with current protocols.

Ethanol-Induced Upregulation of 10-Formyltetrahydrofolate Dehydrogenase Helps Relieve Ethanol-Induced Oxidative Stress

ABSTRACT Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the −124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBPα was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBPα helps relieve embryonic oxidative stress induced by ethanol exposure.

Zebrafish 10-formyltetrahydrofolate dehydrogenase is similar to its mammalian isozymes for its structural and catalytic properties

10-Formyltetrahydrofolate dehydrogenase from zebrafish has been cloned and expressed in both Escherichia coli and yeast. In addition, the N-terminal and C-terminal domains have also been cloned and expressed. Each expressed protein was purified to homogeneity and structural and kinetic properties determined. These studies show that the zebrafish enzyme is structurally and catalytically very similar to the enzymes from mammalian sources, suggesting that zebrafish can be used to study the in vivo function of 10-formyltetrahydrofolate dehydrogenase.

Knocking down 10-formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement

BACKGROUND Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase. METHODS 10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC. RESULTS The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain. CONCLUSIONS 10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis. GENERAL SIGNIFICANCE These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.

Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1

The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys₂-His₂ zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coli Rosetta (DE3) cells as a Nus-His-tag fusion protein. After induction with isopropyl thiogalactoside, the protein was purified with a Ni-Sepharose column, and approximately 5-8 mg of pure protein was obtained per liter of culture. The primary sequence and the predicted partial tertiary structure of the potential recombinant zebrafish Sp1 protein are similar to those of human Sp1. The DNA affinity precipitation assay and dual-luciferase promoter activity assay further confirm the nature of the recombinant zebrafish Sp1 protein as a transcription factor. Our results show that zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1.

Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

Structures of the hydrolase domain of 10-formyltetrahydrofolate dehydrogenase from zebrafish and its complexes are reported.