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.

Following its uptake from outside, in the cell riboflavin undergoes an ATP-dependent phosphorylation catalyzed by riboflavin kinase (RFK, E.C. 2.7.1.26) to form FMN, most of which is further converted by FAD synthase (FADS, E.C. 2.7.7.2) into FAD, the cofactor for many flavoenzymes involved in several biological processes in different cellular compartments. We previously demonstrated that beside a cytosolic enzyme (i.e. isoform 2 of human FLAD1 gene product) a mitochondrial isoform of FADS exists (isoform 1) (1, 2), presumably responsible for the biogenesis of mitochondrial flavoenzymes. Interestingly, the possibility of unexpected localizations for FADS has arisen (3) and, meanwhile, at least two new isoforms of FADS have been deposited in databases. Among FAD-dependent enzymes, lysine-specific demethylase-1 (E.C. 1.14.11.B1) is a nuclear protein recently shown to regulate cellular energy balance depending on FAD availability (4). These observation prompted us to investigate on the presence of flavin coenzymes in the nucleus and on a possible metabolism of FAD in this organelle. To this aim HPLC analysis of acid-precipitable flavins in nuclei isolated from rat liver were performed. The observation of a time-dependent decrease of FAD suggested us the existence of a hydrolytic activity, as confirmed by spectrofluorimetric measurements revealing an increase in fluorescence due to conversion of FAD into its more fluorescent precursor FMN/riboflavin (5). Immunoblotting experiments performed on rat liver nuclei using a home-made antibody against FADS revealed the enrichment of an immunoreactive band in respect to the other sub-cellular fractions. Confocal microscopy on several mammalian cells confirmed the existence of FADS co-localizing with nuclear markers. 1. Barile et al., Eur J Biochem, 2000, 15, 4888-900. 2. Torchetti et al., Mitochondrion, 2010, 10, 263-73. 3. Lin et al., J Neurol, 2009, 256, 774-82. 4. Hino et al., Nat Commun, 2012, 3, 758. 5. Brizio et al., Eur J Biochem, 1997, 3, 777-85.

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors

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 synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS–PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent Km and Vmax values for FMN equal to 1.3 ± 0.7 lM and 4.4 ± 1.3 nmol min1 mg protein1, respectively, and was inhibited by FMN at concentration higher than 1.5 lM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

BACKGROUND/AIM: Riboflavin transport in enterocytes is mediated by three translocators: RFVT3 located on the apical membrane, and RFVT1 and RFVT2 on the basolateral membrane. The aim of this study was to investigate whether the expression levels of RFVTs are altered in human colorectal cancer (CRC). MATERIALS AND METHODS: In human colon adenocarcinoma cell lines (CaCo2, DLD-1, HT-29) and in tissues of patients with CRC, gene and protein expression levels were evaluated by real time-polymerase chain reaction and western blotting. Intracellular flavin content was determined by high-performance liquid chromatography. RESULTS: RFVT3 and RFVT2 gene and protein expression levels were higher in DLD-1 and HT-29 compared to Caco2 cells. In HT-29 cells, the RFVT1 protein level was drastically lower. These differences are presumably responsible for the higher total flavin content in DLD-1 and HT-29 cells. In tumor tissues of patients with CRC, RFVT1 content was reduced at both protein and mRNA levels compared to normal mucosa. RFVT3 and RFVT2 gene expression levels were increased, while protein expression was reduced, with a small reduction in riboflavin amount. CONCLUSION: This study provides first evidence that transcription/translation of RFVTs are profoundly altered in CRC.

The antiserum is directed against human FAD synthase or FMN:ATP adenylyl transferase, a collection of enzymatic isoforms involved in FAD biosynthesis. It specifically reacts with the isoforms in human (and rat) biopsies, cellular extracts, plasma, and blood lysates, liquor. It is able to immune-react with both domains contained in the longest isoforms. FAD synthase deficiency and/or impairment have been linked to certain human neuro-muscular diseases, such as the RR-MADD, which, if early diagnosed, can be treated with the simple ingestion of riboflavin in high doses. Early diagnosis requires measurements of the human enzyme level. Main advantages Methods for measuring FAD synthase activity require specific competences in enzymology and spectrofluorometric assay, thus are time and money consuming. The antibody permits a rapid and simple analysis feasible by any laboratories. Innovative aspects The patented antibody is the best molecular test available. It is of interest for research and diagnostic purposes. It is expected that several research group and companies should purchase the antiserum. Differently from other antisera raised against the same enzyme, our antiserum recognizes each FAD synthase isoform. It has been tested in dot blot, immuno-blotting and immuno-fluorescence analysis. The antiserum also recognizes isoforms from model organisms like S. cerevisiae and C. elegans

A device for use when taking photographs used in the clinical and/or medico-legal and forensic sector, to establish the age of traumatic cutaneous lesions in the form of bruising.

Antibody and method of production of antibodies obtained using as antigen an isoform of the human FAD synthetase, obtained by means of technology of recombinant DNA cloned and over-expressed in microorganisms cultures in particular a colony of E. coli Rosetta (DE3) cells, in which said antigen is purified and concentrated by means of electrophoresis and extraction in tampon before being inoculated in the test animal.