Membrane Proteins: New Approaches to Probes, Technologies, and Drug Design

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Membrane Proteins: New Approaches to Probes, Technologies, and Drug Design, Part II.

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OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics.

OCTN is a small subfamily of membrane transport proteins that belongs to the larger SLC22 family. Two of the three members of the subfamily, namely, OCTN2 and OCTN1, are present in humans. OCTN2 plays a crucial role in the absorption of carnitine from diet and in its distribution to tissues, as demonstrated by the occurrence of severe pathologies caused by malfunctioning or altered expression of this transporter. These findings suggest avoiding a strict vegetarian diet during pregnancy and in childhood. Other roles of OCTN2 are related to the traffic of carnitine derivatives in many tissues. The role of OCTN1 is still unclear, despite the identification of some substrates such as ergothioneine, acetylcholine, and choline. Plausibly, the transporter acts on the control of inflammation and oxidative stress, even though knockout mice do not display phenotypes. A clear role of both transporters has been revealed in drug interaction and delivery. The polyspecificity of the OCTNs is at the base of the interactions with drugs. Interestingly, OCTN2 has been recently exploited in the prodrug approach and in diagnostics. A promising application derives from the localization of OCTN2 in exosomes that represent a noninvasive diagnostic tool

Functional and Molecular Effects of Mercury Compounds on the Human OCTN1 Cation Transporter: C50 and C136 Are the Targets for Potent Inhibition

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ASCT1 and ASCT2: Brother and Sister?

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins.This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences

amino acid transporters in drug discovery

Administered drugs interact with membrane transporters of epithelia, Blood Brain Barrier and other districts influencing their delivery and efficacy. Drugs can also be used as inhibitors of transporters involved in human pathology. Drug-transporter interactions are responsible of off-target effects contributing to toxicity. High Throughput Screening technologies increased the potential applications in therapy or in predicting side effects. These strategies will be helpful in reducing animal experimentation. The identification of transporters important for drug absorption, delivery and side effect production and the best technologies for studying interactions are the main goals in this field. Amino acid transporters are not yet considered in human therapy in spite of their involvement in several pathologies. The function of the amino acid transporters EAAT1, ASCT2, GLYT2, GLYT1, B0AT1, LAT1 and LAT2 is so far well characterized. Some structural data on these transporters have also been obtained by bioinformatics. Interactions of these proteins with several drugs have been well defined at the molecular level. Large scale and, in some cases, high throughput screening of pharmacological compounds make these transporters of particular interest and potential application in human health

Exploiting Cysteine Residues of SLC Membrane Transporters as Targets for Drugs.

The observation that cysteine is the top gainer amino acid during evolution attracted the attention of scientists dealing with protein chemistry. The thiol group of cysteine, indeed, is a potential site for several types of reactions with variable specificity and strength. This feature proved to be promising also in the field of membrane transporters that represent boundary proteins fundamental for cell homeostasis. These proteins are classified, according to the driving force for transport, in primary or secondary active transporters. Another frequently used classification is nowadays based on phylogenesis. Two major groups are identified that take into account both criteria: the ABC and the SLC transporters, the second being much more numerous. The cellular localization of the transporters makes them very attractive for drug design. Moreover, the presence of at least one cysteine residue in all the annotated SLC transporters, so far, highlights the possibility of using the thiol (SH) residue for covalent drug targeting. Even if a delay exists in this research field due to the scarce knowledge of structure/function relationships, the setup of novel experimental tools for studying SLC proteins of plasma and organelle membranes opens an important perspective in pharmacology

The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology

SLC1A5, known as ASCT2, is a neutral amino acid transporter belonging to the SLC1 family and localized in the plasma membrane of several body districts. ASCT2 is an acronym standing for Alanine, Serine, Cysteine Transporter 2 even if the preferred substrate is the conditionally essential amino acid glutamine, with cysteine being a modulator and not a substrate. The studies around amino acid transport in cells and tissues began in the ‘60s by using radiolabeled compounds and competition assays. After identification of murine and human genes, the function of the coded protein has been studied in cell system and in proteoliposomes revealing that this transporter is a Na+ dependent antiporter of neutral amino acids, some of which are only inwardly transported and others are bi-directionally exchanged. The functional asymmetry merged with the kinetic asymmetry in line with the physiological role of amino acid pool harmonization. An intriguing function has been described for ASCT2 that is exploited as a receptor by a group of retroviruses to infect human cells. Interactions with scaffold proteins and post-translational modifications regulate ASCT2 stability, trafficking and transport activity. Two asparagine residues, namely N163 and N212, are the sites of glycosylation that is responsible for the definitive localization into the plasma membrane. ASCT2 expression increases in highly proliferative cells such as inflammatory and stem cells to fulfill the augmented glutamine demand. Interestingly, for the same reason, the expression of ASCT2 is greatly enhanced in many human cancers. This finding has generated interest in its candidacy as a pharmacological target for new anticancer drugs. The recently solved 3D structure of ASCT2 will aid in the rational design of such therapeutic compounds

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

Abstract The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni 2 + -chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [ 3 H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na + . Internal Na + slightly stimulated the antiport. Optimal activity was found at pH 7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥ 70%) and by mercurials and methanethiosulfonates (≥ 80%). Heterologous antiport of [ 3 H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [ 3 H]glutamine/glutamine antiport Km values of 0.097 and 1.8 mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na + on the external side was 32 mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism

Impaired amino acid transport at the blood brain barrier is a cause of autism spectrum disorder

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function