Significance of redox-active cysteines in human FAD synthase isoform 2.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.

FAD synthesis and degradation in the nucleus create a local flavin cofactor pool.

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

Bacterial over-expression and purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain of human FAD synthase: functional characterization and homology modeling.

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble "truncated" form of hFADS lacking the N-terminal domain (Δ(1-328)-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ(1-328)-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ(1-328)-hFADS enzyme shows a slight, but not significant, change of K(m) values (0.24 and 6.23 µM for FMN and ATP, respectively) and of k(cat) (4.2 × 10-2 s-1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ(1-328)-hFADS versus wild-type protein.

Polyunsaturated fatty acids reduce fatty acid synthase and hydroxy-methyl-glutaryl CoA-reductase gene expression and promote apoptosis in HepG2 cell line.

BACKGROUND: n-3 and n-6 polyunsaturated fatty acids (PUFAs) are the two major classes of PUFAs encountered in the diet, and both classes of fatty acids are required for normal human health. Moreover, PUFAs have effects on diverse pathological processes impacting chronic disease, such as cardiovascular and immune disease, neurological disease, and cancer. AIM: To investigate the effects of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) on the proliferation and apoptosis of human hepatoma cell line HepG2 after exposure to increasing concentrations of EPA or ARA for 48 h. Moreover, in the same cells the gene expression of Fatty Acid Synthase (FAS) and 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase (HMG-CoAR) was also investigated. METHOD: Cell growth and apoptosis were assayed by MTT and ELISA test, respectively after cell exposure to increasing concentrations of EPA and ARA. Reverse-transcription and real-time PCR was used to detect FAS and HMG-CoAR mRNA levels in treated cells. RESULTS: Our findings show that EPA inhibits HepG2 cell growth in a dose-dependent manner, starting from 25 µM (P < 0.01, one-way ANOVA test and Dunnett's post test) and exerts a statistically significant pro-apoptotic effect already at 1 µM of EPA. Higher doses of ARA were need to obtain a statistically significant inhibition of cell proliferation and a pro-apoptotic effect in these cells (100 µM, P < 0.01, one-way ANOVA test and Dunnett's post test). Moreover, a down-regulation of FAS and HMG-CoAR gene expression was observed after EPA and ARA treatment in HepG2 cells, starting at 10 µM (P < 0.05, one-way ANOVA test and Dunnett's post test). CONCLUSION: Our results demonstrate that EPA and ARA inhibit HepG2 cell proliferation and induce apoptosis. The down-regulation of FAS and HMG-CoAR gene expression by EPA and ARA might be one of the mechanisms for the anti-proliferative properties of PUFAs in an in vitro model of hepatocellular carcinoma.

Synergic effect of eicosapentaenoic acid and lovastatin on gene expression of HMGCoA reductase and LDL receptor in cultured HepG2 cells.

BACKGROUND: PUFAs are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme catalyzing the conversion of HMGCoA to mevalonate, the rate limiting step in cholesterol biosynthesis. Statins represent a class of drugs that are widely used to treat hypercholesterolemia for their ability to inhibit cholesterol biosynthesis and to up-regulate the synthesis of Low Density Lipoprotein (LDL) receptors in the liver. PUFAs mediate many, if not all, actions of statins and this could be one mechanism by which they lower cholesterol levels. The purpose of this study was to investigate whether combined treatment with Eicosapentaenoic acid (EPA) and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. RESULTS: The combined treatment with EPA and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Moreover, we detected a synergistic effect on the inhibition of cancer cell proliferation obtained by combination of EPA and Lovastatin. CONCLUSIONS: The use of EPA, in combination with low doses of Lovastatin may have potential value in treatment of neoplastic diseases.

Alteration of ROS homeostasis and decreased lifespan in S. cerevisiae elicited by deletion of the mitochondrial translocator FLX1.

This paper deals with the control exerted by the mitochondrial translocator FLX1, which catalyzes the movement of the redox cofactor FAD across the mitochondrial membrane, on the efficiency of ATP production, ROS homeostasis, and lifespan of S. cerevisiae. The deletion of the FLX1 gene resulted in respiration-deficient and small-colony phenotype accompanied by a significant ATP shortage and ROS unbalance in glycerol-grown cells. Moreover, the flx1Δ strain showed H2O2 hypersensitivity and decreased lifespan. The impaired biochemical phenotype found in the flx1Δ strain might be justified by an altered expression of the flavoprotein subunit of succinate dehydrogenase, a key enzyme in bioenergetics and cell regulation. A search for possible cis-acting consensus motifs in the regulatory region upstream SDH1-ORF revealed a dozen of upstream motifs that might respond to induced metabolic changes by altering the expression of Flx1p. Among these motifs, two are present in the regulatory region of genes encoding proteins involved in flavin homeostasis. This is the first evidence that the mitochondrial flavin cofactor status is involved in controlling the lifespan of yeasts, maybe by changing the cellular succinate level. This is not the only case in which the homeostasis of redox cofactors underlies complex phenotypical behaviours, as lifespan in yeasts.

Low levels of lipogenic enzymes in peritumoral adipose tissue of colorectal cancer patients.

Lipoprotein lipase (LPL) is the crucial enzyme for intravascular catabolism of triglyceride-rich lipoproteins. Fatty acid synthase (FAS) is a key anabolic enzyme that catalyzes the terminal steps in the novo biosynthesis of 18:2n-6. The involvement of both LPL and FAS in tumor biology has been widely demonstrated in different studies and to verify whether there are regional differences in the expression of these enzymes in visceral adipose tissue from patients with colorectal cancer might be representative of events which sustain tumor growth. The objective of this study was to evaluate LPL and FAS activity and expression of their genes in adipose tissue adjacent to neoplasia and distant from it from patients operated for colorectal cancer. LPL enzymatic activity was evaluated by a fluorescent method and FAS activity by a radiometer assay. Reverse-transcription and real-time PCR were used to detect mRNA levels of two enzymes. Our findings show a significant reduction in both LPL and FAS gene expression and activity levels in adipose tissue adjacent to tumor lesion compared to those detected in paired tissue distant from neoplasia. These results underline the influence of tumor microenvironment on lipid metabolism in adipose tissue, demonstrating a tumor-induced impairment in the formation and lipid storing capacity of adipose tissue in patients with colorectal cancer.