Inflammation and Organic Cation Transporters Novel (OCTNs).

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.

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.

LAT1 (SLC7A5) catalyzes copper(histidinate) transport switching from antiport to uniport mechanism.

LAT1 (SLC7A5) is one of the most studied membrane transporters due to its relevance to physiology in supplying essential amino acids to brain and fetus, and to pathology being linked to nervous or embryo alterations; moreover, LAT1 over-expression is always associated with cancer development. Thus, LAT1 is exploited as a pro-drug vehicle and as a target for anti-cancer therapy. We here report the identification of a new substrate with pathophysiological implications, i.e., Cu-histidinate, and an unconventional uniport mechanism exploited for the Cu-histidinate transport. Crystals of the monomeric species Cu(His)2 were obtained in our experimental conditions and the actual transport of the complex was evaluated by a combined strategy of bioinformatics, site-directed mutagenesis, radiolabeled transport, and mass spectrometry analysis. The LAT1-mediated transport of Cu(His)2 may have profound implications for both the treatment of copper dysmetabolism diseases, such as the rare Menkes disease, and of cancer as an alternative to platinum-based therapies.

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications.

BACKGROUND: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid ß-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. METHODS: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. RESULTS: Itaconate reacts with the CAC causing irreversible inhibition. Dose-response experiment performed with the native and recombinant protein showed IC50 for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50 decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50 of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.

Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203.

The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.

N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98).

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafficking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafficking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafficking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.

Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins.

Both toxic and physiological effects of CO are mostly caused by well described interactions with heme-groups of proteins. Interactions of CO with non-heme proteins have also been unveiled. Besides interaction of CO with mitochondrial heme containing respiratory complexes, a BK channel and the phosphate carrier which do not contain metal cofactors, have been identified as CO targets. However, the molecular mechanisms of interaction with non-metal-containing proteins are not understood. We show in this work the effect of CO on the mitochondrial carnitine carrier (SLC25A20) using CORM-3, a widely recognized CO releasing compound. CO exerts an inhibitory effect at the micromolar concentration on the transport function of the transporter extracted from treated mitochondria. The effect is due to a single Cys residue, C136 as revealed by mass spectrometry analysis. A computational approach predicted the need for vicinal Asp and Lys residues for the C136 carbonylation to occur. These data demonstrate a novel mechanism of interaction of CO with a protein not containing metal atoms and will enable the prediction of CO targets.

Extracellular Vesicles and Cell Pathways Involved in Cancer Chemoresistance.

Chemoresistance is a pharmacological condition that allows transformed cells to maintain their proliferative phenotype in the presence of administered anticancer drugs. Recently, extracellular vesicles, including exosomes, have been identified as additional players responsible for the chemoresistance of cancer cells. These are nanovesicles that are released by almost all cell types in both physiological and pathological conditions and contain proteins and nucleic acids as molecular cargo. Extracellular vesicles released in the bloodstream reach recipient cells and confer them novel metabolic properties. Exosomes can foster chemoresistance by promoting prosurvival and antiapoptotic pathways, affecting cancer stem cells and immunotherapies, and stimulating drug efflux. In this context, a crucial role is played by membrane transporters belonging to ABC, SLC, and P-type pump families. These proteins are fundamental in cell metabolism and drug transport in either physiological or pathological conditions. In this review, different roles of extracellular vesicles in drug resistance of cancer cells will be explored.

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism.

The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.

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.

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.

ASCT1 and ASCT2: Brother and Sister?

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins.This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences.

The Mitochondrial Carnitine Acyl-carnitine Carrier (SLC25A20): Molecular Mechanisms of Transport, Role in Redox Sensing and Interaction with Drugs.

The SLC25A20 transporter, also known as carnitine acyl-carnitine carrier (CAC), catalyzes the transport of short, medium and long carbon chain acyl-carnitines across the mitochondrial inner membrane in exchange for carnitine. The 30-year story of the protein responsible for this function started with its purification from rat liver mitochondria. Even though its 3D structure is not yet available, CAC is one of the most deeply characterized transport proteins of the inner mitochondrial membrane. Other than functional, kinetic and mechanistic data, post-translational modifications regulating the transport activity of CAC have been revealed. CAC interactions with drugs or xenobiotics relevant to human health and toxicology and the response of the carrier function to dietary compounds have been discovered. Exploiting combined approaches of site-directed mutagenesis with chemical targeting and bioinformatics, a large set of data on structure/function relationships have been obtained, giving novel information on the molecular mechanism of the transport catalyzed by this protein.

Functional Study of the Human Riboflavin Transporter 2 Using Proteoliposomes System.

Riboflavin is essential for cell viability. The biologically active forms of riboflavin, FMN and FAD, participate in many biochemical redox reactions including the metabolism of carbohydrates, amino acids, and lipids. Differently from bacteria, fungi, and plants which synthesize riboflavin, higher organisms have lost the ability to synthesize the vitamin and must absorb it from food and intestinal microflora production. The riboflavin flux through cell membranes occurs via specific transporters belonging to the SLC52 family. Three members of this family have been identified so far which show poor homology with the riboflavin transporters of Saccharomyces cerevisiae or bacteria. Alterations of RFVTs are causative of severe diseases. Indeed, under pathological stress, humans are susceptible of developing riboflavin deficiency. Such a deficiency in pregnancy induces fetus abnormalities, and has been indicated as a risk factor for anemia, cancer, cardiovascular diseases, and neurodegeneration. Moreover, inherited diseases are also of interest; the most well-described is the Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset sensorineural deafness and pontobulbar palsy. Numerous polymorphisms of Slc52a2 and Slc52a3 genes associated with this syndrome have been discovered. In spite of their important metabolic role and their relevance to human health, the riboflavin transporters are still poorly characterized. Bacterial overexpression, purification, and protein reconstitution in liposomes represent an up-to-date methodology for obtaining functional data information. The methodology for reconstituting the RFVT2 into proteoliposomes and performing transport assay is described. These methods will be suitable for investigating the functional defects of the variants of RFVTs associated with human pathologies.

Carnitine Traffic in Cells. Link With Cancer.

Metabolic flexibility is a peculiar hallmark of cancer cells. A growing number of observations reveal that tumors can utilize a wide range of substrates to sustain cell survival and proliferation. The diversity of carbon sources is indicative of metabolic heterogeneity not only across different types of cancer but also within those sharing a common origin. Apart from the well-assessed alteration in glucose and amino acid metabolisms, there are pieces of evidence that cancer cells display alterations of lipid metabolism as well; indeed, some tumors use fatty acid oxidation (FAO) as the main source of energy and express high levels of FAO enzymes. In this metabolic pathway, the cofactor carnitine is crucial since it serves as a "shuttle-molecule" to allow fatty acid acyl moieties entering the mitochondrial matrix where these molecules are oxidized via the ß-oxidation pathway. This role, together with others played by carnitine in cell metabolism, underlies the fine regulation of carnitine traffic among different tissues and, within a cell, among different subcellular compartments. Specific membrane transporters mediate carnitine and carnitine derivatives flux across the cell membranes. Among the SLCs, the plasma membrane transporters OCTN2 (Organic cation transport novel 2 or SLC22A5), CT2 (Carnitine transporter 2 or SLC22A16), MCT9 (Monocarboxylate transporter 9 or SLC16A9) and ATB0, + [Sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) or SLC6A14] together with the mitochondrial membrane transporter CAC (Mitochondrial carnitine/acylcarnitine carrier or SLC25A20) are the most acknowledged to mediate the flux of carnitine. The concerted action of these proteins creates a carnitine network that becomes relevant in the context of cancer metabolic rewiring. Therefore, molecular mechanisms underlying modulation of function and expression of carnitine transporters are dealt with furnishing some perspective for cancer treatment.

Membrane Transporters for Amino Acids as Players of Cancer Metabolic Rewiring.

Cancer cells perform a metabolic rewiring to sustain an increased growth rate and compensate for the redox stress caused by augmented energy metabolism. The metabolic changes are not the same in all cancers. Some features, however, are considered hallmarks of this disease. As an example, all cancer cells rewire the amino acid metabolism for fulfilling both the energy demand and the changed signaling routes. In these altered conditions, some amino acids are more frequently used than others. In any case, the prerequisite for amino acid utilization is the presence of specific transporters in the cell membrane that can guarantee the absorption and the traffic of amino acids among tissues. Tumor cells preferentially use some of these transporters for satisfying their needs. The evidence for this phenomenon is the over-expression of selected transporters, associated with specific cancer types. The knowledge of the link between the over-expression and the metabolic rewiring is crucial for understanding the molecular mechanism of reprogramming in cancer cells. The continuous growth of information on structure-function relationships and the regulation of transporters will open novel perspectives in the fight against human cancers.

Interaction of Cholesterol With the Human SLC1A5 (ASCT2): Insights Into Structure/Function Relationships.

The human SLC1A5 commonly known as ASCT2 is a sodium-dependent neutral amino acid antiporter involved in transmembrane traffic of glutamine that is exchanged through the cell membrane with smaller amino acids such as serine or threonine. Due to the strong overexpression in human cancers, ASCT2 is widely studied for its relevance to human health. Of special interest are the aspects related to the regulation of its function. The role of cholesterol as a modulator of the transport activity has been studied using a combined strategy of computational and experimental approaches. The effect of cholesterol on the Na ex + -[3H]glutamineex/glutaminein antiport in proteoliposomes has been evaluated by adding cholesteryl hemisuccinate. A strong stimulation of transport activity was observed in the presence of 75 µg cholesteryl hemisuccinate per mg total lipids. The presence of cholesterol did not influence the proteoliposome volume, in a wide range of tested concentration, excluding that the stimulation could be due to effects on the vesicles. cholesteryl hemisuccinate, indeed, improved the incorporation of the protein into the phospholipid bilayer to some extent and increased about three times the Vmax of transport without affecting the Km for glutamine. Docking of cholesterol into the hASCT2 trimer was performed. Six poses were obtained some of which overlapped the hypothetical cholesterol molecules observed in the available 3D structures. Additional poses were docked close to CARC/CRAC motifs (Cholesterol Recognition/interaction Amino acid Consensus sequence). To test the direct binding of cholesterol to the protein, a strategy based on the specific targeting of tryptophan and cysteine residues located in the neighborhood of cholesterol poses was employed. On the one hand, cholesterol binding was impaired by modification of tryptophan residues by the Koshland's reagent. On the other hand, the presence of cholesterol impaired the interaction of thiol reagents with the protein. Altogether, these results confirmed that cholesterol molecules interacted with the protein in correspondence of the poses predicted by the docking analysis.

Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli.

BACKGROUND: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. METHODS: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. RESULTS: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. CONCLUSIONS: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

Exploiting Cysteine Residues of SLC Membrane Transporters as Targets for Drugs.

The observation that cysteine is the top gainer amino acid during evolution attracted the attention of scientists dealing with protein chemistry. The thiol group of cysteine, indeed, is a potential site for several types of reactions with variable specificity and strength. This feature proved to be promising also in the field of membrane transporters that represent boundary proteins fundamental for cell homeostasis. These proteins are classified, according to the driving force for transport, in primary or secondary active transporters. Another frequently used classification is nowadays based on phylogenesis. Two major groups are identified that take into account both criteria: the ABC and the SLC transporters, the second being much more numerous. The cellular localization of the transporters makes them very attractive for drug design. Moreover, the presence of at least one cysteine residue in all the annotated SLC transporters, so far, highlights the possibility of using the thiol (SH) residue for covalent drug targeting. Even if a delay exists in this research field due to the scarce knowledge of structure/function relationships, the setup of novel experimental tools for studying SLC proteins of plasma and organelle membranes opens an important perspective in pharmacology.

Human mitochondrial carnitine acylcarnitine carrier: Molecular target of dietary bioactive polyphenols from sweet cherry (Prunus avium L.).

The effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity.