Browsing by Author "Indiveri, Cesare"

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The effect of cholesterol on the function of eukaryotic membrane transporters for amino acids
(Università della Calabria, 2020-11-25) Cosco, Jessica; Cerra, Maria Carmela; Indiveri, Cesare
Amino acid transport in mammalian cells is mediated by different amino acid transporters. Amino acid flow, which is important under physiological conditions, becomes particularly relevant under pathological conditions such as in cancer cells where high demand for these nutrients is required to satisfy the uncontrolled growth and proliferation. Therefore, to guarantee a sufficient supply of nutrients a lot of amino acid transporters are highly expressed in cancer cells. In this context, the amino acid transporters hLAT1 and hASCT2 are widely studied for their role as potential targets for drug development. hASCT2 belongs to SLC1 family and assembles at the plasma membrane as a trimeric complex. Studies conducted using the recombinant protein showed that this transporter is strongly stimulated by cholesterol. The stimulation is due to an improvement of protein insertion in the phospholipid bilayer and direct interaction with the protein. In fact, cholesterol increased the Vmax of the transport, without affecting the external Km, indicating that it increases the rate of conformational changes. Thanks to docking analysis, 6 putative cholesterol binding sites were predicted, some of these matched with the electron densities identified on the cryo-EM structure of ASCT2. Two poses are on the TM6, where a CRAC and a CARC motif has been identified. Experimental demonstrated the direct binding of cholesterol to the protein. In particular, Koshland’s reagent and SH-reagents have been used for the target of tryptophan and cysteine residues close to the cholesterol poses. hLAT1 belongs to SLC7 family and it forms a heterodimer complex (HAT) with the glycoprotein 4F2hc (also known as CD98 in mice), a member of SLC3 family. hLAT1 is the sole component involved in the transport of essential amino acids, as previously demonstrated (Napolitano, Scalise et al. 2015). In this work, the influence of cholesterol has been evaluated on the recombinant protein hLAT1. Moreover, putative regulators involved in energy metabolism have been tested on the transport. The transport activity increased up 75 μg cholesterol/ mg phospholipids. Moreover, the internal substrate affinity increased in the presence of cholesterol suggesting a stabilization of the inward conformation of hLAT1. The transporter is also stimulated by ATP at physiological concentration. This effect occurs only in the presence of cholesterol and was seen also on the native protein. This finding suggested that cholesterol and ATP binding sites are close to each other. The computational analysis confirmed this hypothesis. In fact, a hydrophobic region between the TMs 1, 5 and 7 was found to be close to a hydrophilic one. Docking results for ATP suggested an electrostatic interaction of the ϒ-phosphate of ATP with Lys 204, which was confirmed by site-directed mutagenesis. This residue is conserved in the other SLC7 proteins and for a serendipity event, it has been seen that Lys204 is also important in the substrate binding and pH-sensitive. In this work, the attention was focused also on another amino acid transporter CAT2, from Solanum lycopersicum. The specific interest in tomato resides in the well-recognized role for this species in biotechnology. In fact, tomato has been used as the primary model for the study of climacteric fruit ripening. SlCAT2 belongs to APC superfamily, as LAT1, and it is involved in the transport of cationic amino acids like arginine, lysine and the non-proteogenic amino acid ornithine. The experimental data on CAT2 highlighted an asymmetric regulation by cations and osmotic pressure, in line with the localization of the transporter in vacuoles. Like the other human transporters, CAT2 is also stimulated by cholesterol. On the basis of the 3D structure of the amino acid transporter GkApcT, the homology model of SlCAT2 was built and putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of membrane transporters to metabolic and membrane changes. Moreover the identification of hydrophobic or hydrophilic sites interacting with cholesterol or physiological effectors, respectively, could be important for applications in human pathology.
Il trasportatore di carnitina OCTN2: espressione ed implicazioni fisiopatologiche
(2010) Scalise, Mariafrancesca; Indiveri, Cesare; Sisci, Diego
In the present work we studied the plasma membrane transporter of carnitine OCTN2. We firstly investigated the interaction of this transporter with a commonly used drug, the omeprazolo. The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstituted carnitine transporter catalysed a first-order antiport reaction of carnitine with itself or other substrates stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. Omeprazole externally added to the proteoliposomes, inhibited the carnitine/carnitine antiport catalysed by the reconstituted transporter. The inhibition was reversed by the treatment of the proteoliposomes by DTE indicating that the inhibition was caused by the formation of omeprazole-transporter mixed disulphide. However, omeprazole caused inhibition of the transport also in the presence of DTE, indicating a second inhibition mechanism of non-covalent nature. The presence of the substrate during the incubation of the omeprazole with the proteoliposomes increased the formation of the mixed-disulphide. Omeprazole did not inhibited when present in the internal proteoliposomal compartment, indicating that the inhibition was specifically due to interaction with the external residues or sites of the protein. Omeprazole was not found to be transported by OCTN2: the structure of the activated form of omeprazole, indeed, possess a charged pyridine group and a methoxyl oxygen at a reciprocal distance of three carbon atoms which fulfil the requirement for interaction with the active site and inhibition but not for transport. The implication of the two mechanism of inhibition of the carnitine transporter in physiopathology may be relevant in the light of the omeprazole concentration reached in the blood after administration: the inhibition by omeprazole, indeed, may lead to a carnitine deficiency-like syndrome more or less evident, depending on the dose and on the individual capacity of metabolizing the drug. In the present work we studied, also, the human isoform of OCTN2: (i) we obtained the heterologous over-expression and the purification of hOCTN2 in a bacterial host, E. coli and (ii) we evaluated the expression profile of hOCTN2 in different cancer cell lines and in keratinocytes retrotransduced with HPV16 E6E7. The cDNA coding for hOCTN2 was cloned in two bacterial expression vector and, following two different strategies, we over-expressed the hOCTN2 protein as fusion protein with GST and as hOCTN2 without any tag but with codon bias. In both cases the protein was present in the insoluble fraction of induced lysate and was, then, solubilized with a ionic detergent, Sarkosyl, together with chaotropic agent, Urea. After solubilization the protein was purified on Nichel-chelating chromatography column, obtaining the protein solubilized in Triton X- 100. In the present work we described the expression profile of hOCTN2 in different cancer cell lines and in keratinocytes immortalized by HPV16E6E7: we observed a down-regulation at mRNA levels of hOCTN2 which was reversed by treating cells with 5-aza-cytidine a DNA demetilating agent. Cells over- expressing hOCTN2 were more sensitive to the chemotherapic agent cisplatin. This observation is a preliminary result suggesting a potential role of hOCTN2 in carcinogenesis or in cancer development.
Modelli sperimentali cellulari e molecolari per la valutazione della tossicità di xenobiotici
(2013-12-02) Peta, Valentina; Sisc, Diego; Indiveri, Cesare
The aim of my thesis was to find two different experimental models to study cellular and molecular xenobiotics toxicity. In the first part of the present work we studied the interaction between two different plasma membrane transporters (OCTN2 and OCTN1), mercury reagents and heavy metals. Mercury and heavy metals in general cause toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes containseveral Cys residues. One of the most up to date method of studying transport is the reconstitution of transportersin proteoliposomes. This method has been used as a useful approach to test the effect of HgCl2, methylmercury(MeHg) and Cadmium on the carnitine (OCTN2) transporter, extracted from rat kidney brush border membranes and reconstituted in liposomes by removing the detergent withhydrophobic chromatography columns, and the human organic cation transporter (OCTN1) overexpressed in E. coli, purified by Ni-chelating chromatography and reconstituted in liposomes by detergent removal with a batch-wise procedure.Transport was measured as [3H]carnitine uptake into proteoliposomescontaining carnitine (antiport reaction) in the case of rat OCTN2 and as [14C]tetraethylammonium uptake in the case of human OCTN1. Mercurials and heavy metals strongly inhibited the transport. Inhibition was reversed by1,4-dithioerythritol (DTE), L-cysteine (Cys), and N-acetyl-Lcysteine (NAC) indicating that it was caused by covalent reactionof mercurials and heavy metals with Cys residue(s) of OCTN2 and OCTN1.The presence of substrate prevented the inhibition in rat OCTN2 transporter indicating that the mercurial binding residue (Cys) is in the substrate binding site. No substrate protection was found in the case of the human OCTN1, so probably mercurial and heavy metal binding residue is away from substrate binding site. For the human OCTN1 we also tested the effect of chemical reagents which are known to form mixeddisulphides with proteins SH residues, MTS reagents. MTSEA exerted inhibition of transport very similar to those observed for the heavy metals and as the toxic compounds. To ascertain the involvement of Cys residues in the interaction of the human OCTN1 with the xenobiotics and to identify the possible target of the reagents, 7 mutants were prepared in which the seven cysteines present in the transmembrane domains or in the extracellular loop were mutated to alanine. An additional mutant lacking two Cys residues has also been constructed (C50A/C136A). We studies the dose-response curves of the transporter for each inhibitor, the mercury reagents showed similar behavior, both HgCl2 and MeHg strongly inhibited the WT and the mutants C81A, C113A, C236A, C270A and C374A. While a clear shift of the curves towards higher concentrations of the compounds was observed in the case of mutants C50A and C136A indicating a decrease of affinity of these mutants for the mercury reagents. The experiment on the double mutant C50A/C136A showed a nearly complete lack of inhibition by the two reagents demonstrating the two Cys residues were indeed the target of the mercury compounds. The homology model of the human OCTN1 confirms the experimental data obtained in this work, in fact the model shows that the two cysteine residues (Cys-50 and Cys-136) are exposed to the extracellular site of the plasma membrane and are accessible to sulfhydryl groups reagents. In the present work we studied, also, biomarkers expression and nephrotoxic effect induced by drugs in human kidney primary cells model. Kidney is a primary target of drug-induced toxicity. Toxic effects on the kidney related to drugs are both common and expected, given the kidney's roles in plasma filtration and maintenance of metabolic homeostasis. As such, glomerular, tubular and renal interstitial cells frequently are exposed to concentrations of drugs, which can induce changes in kidney function and structure. We tested three different drugs: 2-Bromoethanamine an analgesic, cisplatin a chemotherapeutic agent and cyclosporine a immunosuppressive agent. We found that after the addition of these drugs on human primary kidney cells there is an increase in the expression of two different biomarkers: Osteopontin (OPN) a distal tubule biomarker and KIM-1 a membrane glycoprotein expressed by proximal tubule cells after kidney injury. We also found the toxic concentrations after 24 hours of exposition to the three different drugs, we quantified the percentual of necrotic and apoptotic cells and we studied the toxicological effect of these drugs on cellular organelles like mitochondria. In conclusion proteoliposomes represent a suitable molecular model for studying the interaction of plasma membrane transport and toxic compounds, such as mercurials, and human primary cell culture is a valuable tool to study cell toxicity mechanisms of different drugs. Both the experimental models are a novel and potentially important tools in drug discovery and in the understanding of toxicity mechanism of xenobiotics compounds.
Structure/function relationships of the human heterodimeric amino acids transporter 4F2hc/LAT1
(2017-06-09) Napolitano, Lara; Canonaco, Marcello; Indiveri, Cesare; Scalise, Mariafrancesca
Amino acids transport in mammalian cells is mediated by different amino acid transporters whose activity allow the flow of an important source for metabolic need of cells. Moreover, some amino acids such as Gln, Arg and Leu work as signalling molecules and their availability and concentration represent key factors in the regulation of intracellular signalling pathways responsible of cellular growth. Thus, amino acids flow, which is important under physiological condition, becomes particularly relevant under pathological conditions such as in tumours cells to satisfy their unique metabolic and proliferative needs. Therefore, since in tumours upregulation of amino acids transporters is an important step to satisfy the increased demand for these nutrients, the same transporters are potential drug targets for cancer therapy. However, the certainty that a specific transporter could be a target in human therapy requires its functional characterization and the knowledge of the enchanting structure/function relationships. In this context, an important transporter that became of particular interest for its overexpression in many tumours is LAT1, and the aim of this work has been that to shed light on still unclear aspects of its function hLAT1 belongs to SLC7 family and into the plasma membrane forms heterodimers with the glycoprotein 4F2hc (also known as CD98 in mice), member of SLC3 family. Studies conducted in intact cells showed that 4F2hc/LAT1 complex catalyses amino acids transport; however, in this experimental model it was not possible to clarify whether one or both subunits are competent for transport activity and substrate recognition. Thus, aimed to unravel the dark side of 4F2hc/LAT1 mediated transport, different experimental strategies were adopted allowing to demonstrate that LAT1 is the sole transport competent unit of the heterodimer. Indeed, using western blot analyses and transport assays in liposomes reconstituted with proteins extracted from SiHa cells and in liposomes reconstituted with recombinant LAT1, it has been demonstrated that neither the covalent interaction nor the association of 4F2hc with LAT1 influence transport and specificity of LAT1. Moreover, the suitability of proteoliposome model used for reconstitution of recombinant LAT1, allowed to identify a functional asymmetry of this transporter which, on a physiological point of view, exhaustively elucidates the reciprocal correlation between the transport activity of LAT1 and that of another important amino acids transporter overexpressed in tumours cells, ASCT2. To the same extent, proteoliposome tool together with bioinformatics and site-directed mutagenesis have been useful to probe critical residues of the substrate binding site of LAT1. These results laid the groundwork for deciphering molecular mechanism of LAT1 function and for setting up studies aimed to identify new potent and specific inhibitors great for human health.