Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark.

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

Lysine 204 is crucial for the antiport function of the human LAT1 transporter.

LAT1 (SLC7A5) catalyzes an antiport reaction of amino acids with specificity towards the essential ones. It is mainly expressed at the Blood Brain Barrier and placenta barriers, but it becomes over-expressed in virtually all human cancers even if originating from tissues with lower expression levels. The antiport reaction of LAT1 is crucial at the BBB since its inherited loss causes Autism Spectrum Disorder. We have investigated the possible molecular determinant of the antiport by site-directed mutagenesis, in vitro transport assay and computational analysis. Previous data indicated that mutation of K204 impairs, but does not knock-out LAT1 functionality. We have investigated the activity changes in the K204Q mutant by following the transport of [3H]-histidine, one of the major substrates, in proteoliposomes harbouring the WT or K204Q. In the mutant, the [3H]-histidine uptake and efflux are not more stimulated by the counter-substrate as they occur in the WT. Moreover, the mutation strongly decreases the substrate affinity and alters the pH dependence of K204Q. Molecular Dynamics analysis correlates well with the experimental data since it shows that substrate prematurely escapes the binding site. In addition, the K204Q shows a strongly increased mobility in those regions, transmembrane domains and random coils, involved in the transport cycle. The identified Lys residue could be responsible of the same phenomenon in those members of the SLC7 family, described as antiporters, in which it is conserved.

LAT1 (SLC7A5) catalyzes copper(histidinate) transport switching from antiport to uniport mechanism.

LAT1 (SLC7A5) is one of the most studied membrane transporters due to its relevance to physiology in supplying essential amino acids to brain and fetus, and to pathology being linked to nervous or embryo alterations; moreover, LAT1 over-expression is always associated with cancer development. Thus, LAT1 is exploited as a pro-drug vehicle and as a target for anti-cancer therapy. We here report the identification of a new substrate with pathophysiological implications, i.e., Cu-histidinate, and an unconventional uniport mechanism exploited for the Cu-histidinate transport. Crystals of the monomeric species Cu(His)2 were obtained in our experimental conditions and the actual transport of the complex was evaluated by a combined strategy of bioinformatics, site-directed mutagenesis, radiolabeled transport, and mass spectrometry analysis. The LAT1-mediated transport of Cu(His)2 may have profound implications for both the treatment of copper dysmetabolism diseases, such as the rare Menkes disease, and of cancer as an alternative to platinum-based therapies.

The Nutraceutical Alliin From Garlic Is a Novel Substrate of the Essential Amino Acid Transporter LAT1 (SLC7A5).

The plasma membrane transporter LAT1 (SLC7A5) is a crucial player for cell homeostasis because it is responsible for providing cells with essential amino acids and hormones. LAT1 forms a functional heterodimer with the cell surface antigen heavy chain CD98 (also known as 4F2hc and SLC3A2), a type II membrane glycoprotein, which is essential for LAT1 stability and localization to the plasma membrane. The relevance of LAT1 for human metabolism is also related to its altered expression in human diseases, such as cancer and diabetes. These features boosted research toward molecules that are able to interact with LAT1; in this respect, the recent resolution of the LAT1-CD98 3D structure by Cryo-EM has opened important perspectives in the study of the interaction with different molecules in order to identify new drugs to be used in therapy or new substrates of natural origin to be employed as adjuvants and food supplements. In this work, the interaction of LAT1 with alliin, a garlic derivative, has been investigated by using a combined approach of bioinformatics and in vitro transport assays. Alliin is a nutraceutical that has several beneficial effects on human health, such as antidiabetic, anticarcinogenic, antioxidant, and anti-inflammatory properties. The computational analysis suggested that alliin interacts with the substrate binding site of LAT1, to which alliin was docked. These data were then confirmed by the competitive type inhibition measured in proteoliposomes. Interestingly, in the same experimental model, alliin was also revealed to be a substrate of LAT1.

N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98).

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafficking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafficking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafficking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.