Structure/function relationships of the human heterodimeric amino acids transporter 4F2hc/LAT1
View/ Open
Creato da
Napolitano, Lara
Canonaco, Marcello
Indiveri, Cesare
Scalise, Mariafrancesca
Metadata
Show full item recordDescrizione
Format
/
Dottorato di Ricerca in Scienze della Vita. Ciclo XXIX SSD; 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.; Università della Calabria.Soggetto
Amino Acid; Protein transpor
Relazione
BIO/10;