Dipartimento di Biologia, Ecologia e Scienze della Terra

Now showing 1 - 2 of 2

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.
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.

Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato

Browse

Filters

Settings

Sort By

Results per page

Search Results