Proteoliposomes as Tool for Assaying Membrane Transporter Functions and Interactions with Xenobiotics

Abstract

Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs. Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery.

A Call for Systematic Research on Solute Carriers

“…cells (Cong et al, 2013;Qi et al, 2013) Chemical genomics high-throughput phenotypic screening (Carette et al, 2009;Reiling et al, 2011;Winter et al, 2014) SLC interactome label-free high-throughput AP-MS; BirA-mediated BioID; membrane interaction mapping (Cox and Mann, 2011;Lambert et al, 2015;Petschnigg et al, 2014;Varjosalo et al, 2013) Metabolomic data and SLC genetic polymorphisms genetic association studies; populationwide whole-genome sequencing; rare disease genome sequencing coupled with deep metabolomics (Shin et al, 2014) Metabolome-wide transport assays, in dependence of individual SLC gene alteration high-throughput accurate LC/ GC-mass spectrometry and databases; libraries of metabolites; k.o. cells (Kell, 2004) Transport assays using recombinant proteins proteoliposomes; liposome microarrays; pure solutes, complex body fluids (Krumpochova et al, 2012;Saliba et al, 2014;Scalise et al, 2013) High-throughput determination of 3D structure single-particle cryo-EM; high-throughput crystallization protocols; serial femtosecond crystallography (Bai et al, 2015;Bartesaghi et al, 2015;Chapman et al, 2011;Moraes et al, 2014;Zeev-Ben-Mordehai et al, 2014;Zhou et al, 2015) Potent and selective chemical probes for each SLC better libraries; more accurate screening technologies; assays to assess target engagement and specificity in cells and tissues (Edwards et al, 2009;Frye, 2010) 2,500. To allow the interrogation of the full data set beyond what can be reasonably visualized in a single network, we include an extended list of SLC co-expression pairs across all tissues ( Table S2).…”

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR

“…Unilamellar lipid vesicles of varying sizes are frequently used to mimic biological membranes in studies of protein-lipid or peptide-lipid interactions (Scalise et al 2013; Mandal et al 2015; Stepanyants et al 2015; LeBarron and London 2016; Veshaguri et al 2016). Unilamellar vesicles expose a maximum amount of membrane surface for binding by peripherally binding membrane proteins (Mandal et al 2015) or antimicrobial peptides (Porcelli et al 2013).…”

Glutamine Transport and Mitochondrial Metabolism in Cancer Cell Growth

“…Being irreversible, covalent inhibitors should be in principle, more efficient in chemically knocking-out the transporters. This strategy has the advantage of facilitating the compound screening studying the effects on the sole target protein, without interferences deriving from other systems present in the whole cells ( 95 ). Then, we identified potent covalent inhibitors of the rat ASCT2 ( 96 ).…”