Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder.

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.

TMBIM5 is the Ca2+ /H+ antiporter of mammalian mitochondria.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

Bacterial over-production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands.

BACKGROUND: SLC38A2 is a ubiquitously expressed Na+-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy. METHODS AND RESULTS: The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane. CONCLUSIONS: The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.

Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes.

The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C12E8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na+-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.

Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter.

BACKGROUND: Escherichia coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies. METHODS AND RESULTS: The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state. CONCLUSIONS: Using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.

OCTN1: A Widely Studied but Still Enigmatic Organic Cation Transporter Linked to Human Pathology and Drug Interactions.

The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives.

Ten percent of human genes encode for membrane transport systems, which are key components in maintaining cell homeostasis. They are involved in the transport of nutrients, catabolites, vitamins, and ions, allowing the absorption and distribution of these compounds to the various body regions. In addition, roughly 60% of FDA-approved drugs interact with membrane proteins, among which are transporters, often responsible for pharmacokinetics and side effects. Defects of membrane transport systems can cause diseases; however, knowledge of the structure/function relationships of transporters is still limited. Among the expression of hosts that produce human membrane transport systems, E. coli is one of the most favorable for its low cultivation costs, fast growth, handiness, and extensive knowledge of its genetics and molecular mechanisms. However, the expression in E. coli of human membrane proteins is often toxic due to the hydrophobicity of these proteins and the diversity in structure with respect to their bacterial counterparts. Moreover, differences in codon usage between humans and bacteria hamper translation. This review summarizes the many strategies exploited to achieve the expression of human transport systems in bacteria, providing a guide to help people who want to deal with this topic.

SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.

Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark.

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1).

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

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.

Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding.

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT "supermutant" of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

Regulatory Aspects of the Vacuolar CAT2 Arginine Transporter of S. lycopersicum: Role of Osmotic Pressure and Cations.

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli and reconstituted in proteoliposomes for transport assay as [³H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K⁺, Na⁺, and Mg2+ present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity; differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.

Discovery of Potent Inhibitors for the Large Neutral Amino Acid Transporter 1 (LAT1) by Structure-Based Methods.

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure-activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-µM range.

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L-1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min-1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

Glutamine transport. From energy supply to sensing and beyond.

Glutamine is the most abundant amino acid in plasma and is actively involved in many biosynthetic and regulatory processes. It can be synthesized endogenously but becomes "conditionally essential" in physiological or pathological conditions of high proliferation rate. To accomplish its functions glutamine has to be absorbed and distributed in the whole body. This job is efficiently carried out by a network of membrane transporters that differ in transport mechanisms and energetics, belonging to families SLC1, 6, 7, 38, and possibly, 25. Some of the transporters are involved in glutamine traffic across different membranes for metabolic purposes; others are involved in specific signaling functions through mTOR. Structure/function relationships and regulatory aspects of glutamine transporters are still at infancy. In the while, insights in involvement of these transporters in cell redox control, cancer metabolism and drug interactions are arising, stimulating basic research to uncover molecular mechanisms of transport and regulation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Bacterial production and reconstitution in proteoliposomes of Solanum lycopersicum CAT2: a transporter of basic amino acids and organic cations.

KEY MESSAGE: The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [3H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [3H]Ornithine and [3H]Lys was also detected at lower efficiency with respect to [3H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

Novel insights into the transport mechanism of the human amino acid transporter LAT1 (SLC7A5). Probing critical residues for substrate translocation.

BACKGROUND: LAT1 (SLC7A5) is the transport competent unit of the heterodimer formed with the glycoprotein CD98 (SLC3A2). It catalyzes antiport of His and some neutral amino acids such as Ile, Leu, Val, Cys, Met, Gln and Phe thus being involved in amino acid metabolism. Interestingly, LAT1 is over-expressed in many human cancers that are characterized by increased demand of amino acids. Therefore LAT1 was recently acknowledged as a novel target for cancer therapy. However, knowledge on molecular mechanism of LAT1 transport is still scarce. METHODS: Combined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes, have been adopted to unravel dark sides of human LAT1 structure/function relationships. RESULTS: It has been demonstrated that residues F252, S342, C335 are crucial for substrate recognition and C407 plays a minor role. C335 and C407 cannot be targeted by SH reagents. The transporter has a preferential dimeric structure and catalyzes an antiport reaction which follows a simultaneous random mechanism. CONCLUSIONS: Critical residues of the substrate binding site of LAT1 have been probed. This site is not freely accessible by molecules other than substrate. Similarly to LeuT, K+ has some regulatory properties on LAT1. GENERAL SIGNIFICANCE: The collected data represent a solid basis for deciphering molecular mechanism underlying LAT1 function.

Riboflavin transport and metabolism in humans.

Recent studies elucidated how riboflavin transporters and FAD forming enzymes work in humans and create a coordinated flavin network ensuring the maintenance of cellular flavoproteome. Alteration of this network may be causative of severe metabolic disorders such as multiple acyl-CoA dehydrogenase deficiency (MADD) or Brown-Vialetto-van Laere syndrome. A crucial step in the maintenance of FAD homeostasis is riboflavin uptake by plasma and mitochondrial membranes. Therefore, studies on recently identified human plasma membrane riboflavin transporters are presented, together with those in which still unidentified mitochondrial riboflavin transporter(s) have been described. A main goal of future research is to fill the gaps still existing as for some transcriptional, functional and structural details of human FAD synthases (FADS) encoded by FLAD1 gene, a novel "redox sensing" enzyme. In the frame of the hypothesis that FADS, acting as a "FAD chaperone", could play a crucial role in the biogenesis of mitochondrial flavo-proteome, several basic functional aspects of flavin cofactor delivery to cognate apo-flavoenzyme are also briefly dealt with. The establishment of model organisms performing altered FAD homeostasis will improve the molecular description of human pathologies. The molecular and functional studies of transporters and enzymes herereported, provide guidelines for improving therapies which may have beneficial effects on the altered metabolism.