A New Frontier for Drug-Coated Balloons: Treatment of "De Novo" Stenosis in Large Vessel Coronary Artery Disease.

Background: Drug-coated balloons (DCB) are a well-established option for treating in-stent restenosis endorsed by European Guidelines on myocardial revascularization. However, in recent years, a strategy of "leaving nothing behind" with DCB in de novo coronary stenosis has emerged as an appealing approach. Methods: We performed a systematic review to evaluate the current literature on the use of drug-coated balloons in the treatment of de novo stenosis in large vessel disease. Results: Observational studies, as well as randomized studies, demonstrated the safety of DCB percutaneous coronary interventions (PCI) in large vessel disease. The rate of major adverse cardiac events is even lower compared to drug-eluting stents in stable coronary artery disease. Conclusions: DCB PCI is feasible in large vessel disease, and future large, randomized studies are ongoing to confirm these results.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs.

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

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.

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.

The involvement of sodium in the function of the human amino acid transporter ASCT2.

Alanine, serine, cysteine transporter 2 (ASCT2) is a membrane amino acid transporter with relevance to human physiology and pathology, such as cancer. Notwithstanding, the study on the ASCT2 transport cycle still has unknown aspects, such as the role of Na+ in this process. We investigate this issue using recombinant hASCT2 reconstituted in proteoliposomes. Changes in the composition of purification buffers show the crucial role of Na+ in ASCT2 functionality. The transport activity is abolished when Na+ is absent or substituted by Li+ or K+ in purification buffers. By employing a Na+ fluorometric probe, we measured an inwardly directed flux of Na+ and, by combining fluorometric and radiometric assays, determined a 2Na+ : 1Gln stoichiometry. Kinetics of Na+ transport suggest that pH-sensitive residues are involved in Na+ binding/transport. Our results clarify the role of Na+ on human ASCT2 transporter activity.

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.

ATP modulates SLC7A5 (LAT1) synergistically with cholesterol.

The plasma membrane transporter hLAT1 is responsible for providing cells with essential amino acids. hLAT1 is over-expressed in virtually all human cancers making the protein a hot-spot in the fields of cancer and pharmacology research. However, regulatory aspects of hLAT1 biology are still poorly understood. A remarkable stimulation of transport activity was observed in the presence of physiological levels of cholesterol together with a selective increase of the affinity for the substrate on the internal site, suggesting a stabilization of the inward open conformation of hLAT1. A synergistic effect by ATP was also observed only in the presence of cholesterol. The same phenomenon was detected with the native protein. Altogether, the biochemical assays suggested that cholesterol and ATP binding sites are close to each other. The computational analysis identified two neighboring regions, one hydrophobic and one hydrophilic, to which cholesterol and ATP were docked, respectively. The computational data predicted interaction of the ϒ-phosphate of ATP with Lys 204, which was confirmed by site-directed mutagenesis. The hLAT1-K204Q mutant showed an impaired function and response to ATP. Interestingly, this residue is conserved in several members of the SLC7 family.

The Human SLC1A5 Neutral Amino Acid Transporter Catalyzes a pH-Dependent Glutamate/Glutamine Antiport, as Well.

ASCT2 is a neutral amino acid transporter, which catalyzes a sodium-dependent obligatory antiport among glutamine and other neutral amino acids. The human ASCT2 over-expressed in Pichia pastoris and reconstituted in proteoliposomes has been employed for identifying alternative substrates of the transporter. The experimental data highlighted that hASCT2 also catalyzes a sodium-dependent antiport of glutamate with glutamine. This unconventional antiport shows a preferred sidedness: glutamate is inwardly transported in exchange for glutamine transported in the counter direction. The orientation of the transport protein in proteoliposomes is the same as in the cell membrane; then, the observed sidedness corresponds to the transport of glutamate from the extracellular to the intracellular compartment. The competitive inhibition exerted by glutamate on the glutamine transport together with the docking analysis indicates that the glutamate binding site is the same as that of glutamine. The affinity for glutamate is lower than that for neutral amino acids, while the transport rate is comparable to that measured for the asparagine/glutamine antiport. Differently from the neutral amino acid antiport that is insensitive to pH, the glutamate/glutamine antiport is pH-dependent with optimal activity at acidic pH on the external (extracellular) side. The stimulation of glutamate transport by a pH gradient suggests the occurrence of a proton flux coupled to the glutamate transport. The proton transport has been detected by a spectrofluorometric method. The rate of proton transport correlates well with the rate of glutamate transport indicating a 1:1 stoichiometry H+: glutamate. The glutamate/glutamine antiport is also active in intact HeLa cells. On a physiological point of view, the described antiport could have relevance in some districts in which a glutamate/glutamine cycling is necessary, such as in placenta.

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.