Molecular characterization, evolutionary and phylogenetic analyses of rice ACT/BAT-type amino acid transporters.

The Amino Acid-Polyamine-Organocation (APC) family transporters containing BAT/ACT (amino acid/choline transporters), PHS/LAT (polyamine H+-symporters) and CAT (cationic amino acid transporters) type transporters are ubiquitously reported in plants. ACT (containing BAT genes) which mediates bidirectional amino acid transport has been poorly characterized till date. In the present study, bioinformatics-based analytical studies have been performed to characterize the structural, functional, and evolutionary features of seven ACT/BAT transporters in the model crop Oryza sativa. Sequence homologies with allied transporters suggest that these seven transporter proteins can also have important roles in regulating amino acid transportation. Evolutionary and phylogenetic study of OsBATs proteins using different statistical models based on CDS, Protein, and UTR regions have brought forward some valuable information regarding their way and patterns of diversification over the time, possibly due to the influence of their prevailing growth conditions. Computational analyses based on promoter regions, exon-intron structural patterns, conserved motifs, secondary and tertiary structures and ligand binding sites with probable ligand names provided clues on their evolutionary structural diversification indicating changes in their functional specification probably needed to combat with the changing environment.

HATs meet structural biology.

Heteromeric amino acid transporters (HATs) are one of the ten types of amino acid transporters present in the human body. Growing interest in the pathophysiological role of this group of transporters in rare and complex diseases and cancer has brought about the recent resolution of various structures of human HATs and bacterial homologues at atomic level. This knowledge sheds light on the mechanisms of transport used by these molecules. Here, we discuss the molecular bases underlying substrate specificity, binding asymmetry, and the impact of disease-causing mutations on transporter biogenesis and function.

The Ectodomains of rBAT and 4F2hc Are Fake or Orphan α-Glucosidases.

It is known that 4F2hc and rBAT are the heavy subunits of the heteromeric amino acid transporters (HATs). These heavy subunits are N-glycosylated proteins, with an N-terminal domain, one transmembrane domain and a bulky extracellular domain (ectodomain) that belongs to the α-amylase family. The heavy subunits are covalently linked to a light subunit from the SLC7 family, which is responsible for the amino acid transport activity, forming a heterodimer. The functions of 4F2hc and rBAT are related mainly to the stability and trafficking of the HATs in the plasma membrane of vertebrates, where they exert the transport activity. Moreover, 4F2hc is a modulator of integrin signaling, has a role in cell fusion and it is overexpressed in some types of cancers. On the other hand, some mutations in rBAT are found to cause the malfunctioning of the b0,+ transport system, leading to cystinuria. The ectodomains of 4F2hc and rBAT share both sequence and structure homology with α-amylase family members. Very recently, cryo-EM has revealed the structure of several HATs, including the ectodomains of rBAT and 4F2hc. Here, we analyze available data on the ectodomains of rBAT and 4Fhc and their relationship with the α-amylase family. The physiological relevance of this relationship remains largely unknown.

Quantitative modelling of amino acid transport and homeostasis in mammalian cells.

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.

A conformational change in the N terminus of SLC38A9 signals mTORC1 activation.

mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.

Millisecond dynamics of an unlabeled amino acid transporter.

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.

Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.

The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

Bifunctional and Unusual Amino Acid ß- or γ-Ester Prodrugs of Nucleoside Analogues for Improved Affinity to ATB0,+ and Enhanced Metabolic Stability: An Application to Floxuridine.

Floxuridine (FUdR, 5-fluoro-2-deoxyuridine) was widely used in patients with tumor. But the poor activity and severe side effects have been observed in the clinic, which resulted from increased degradation cleavage of FUdR to 5-FU by thymidine phosphorylase and reduced transporter-mediated entry into cells. In this study, we have synthesized a series of l-aspartic acid ß-esters and l-glutamic acid γ-esters of FUdR to improve the metabolic stability of FUdR and target FUdR to cancer cells via amino acid transporter ATB0,+ which was exclusively up-regulated in some cancerous tissue. The uptake mechanism, stability, in vitro/in vivo antiproliferation action, pharmacokinetics, and tissue distribution were studied. The combined results showed the unusual 5'-ß-l-Asp-FUdR possessed a better tumor inhibition rate and a better metabolic stability than FUdR through a ATB0,+-mediated prodrug approach. The present study provided the first proof-of-concept of exploiting ATB0,+ for tumor-selective delivery of nucleoside analogues in the form of prodrug.

Genome-wide survey of the amino acid transporter gene family in wheat (Triticum aestivum L.): Identification, expression analysis and response to abiotic stress.

Amino acid transporters (AATs), which transport amino acids across cell membranes, play important roles in alleviating plant damage under stresses and in plant growth. To data, little is known about the AAT genes in wheat because of its complex genome. In this study, a total of 296 AAT genes were identified from the latest wheat genome sequence (IWGSC v1.1) and classified into 12 distinct subfamilies based upon their sequence composition and phylogenetic relationship. The expansion of the wheat AAT family was mainly the results of whole-genome duplication (WGD) and tandem events. The unequal expansion of different subfamilies brought new features to TaAATs. TaAATs were highly expressed and exhibited distinct expression patterns in different tissues. On the basis of homology and expression pattern analysis, we identified several wheat AAT family members that may affect grain quality. In addition, TaAAP3, TaATLa2 and TaATLb13 exhibited sustained expression in response to drought and high-temperature stress. These genes are involved in the response of wheat to abiotic stress by regulating the transport and distribution of amino acids. Overall, our results help to understand the complexity of TaAATs and provide a theoretical basis for further identification and utilization of AATs in wheat and other crop species.

Expression, purification and oligomerization of the S-adenosylmethionine transporter.

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.

Genome-wide identification, characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis).

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1-19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, -6, -12, -13 and - 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems-the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues GltPh and GltTk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner.

Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.

Free-Energy Simulations Resolve the Low-Affinity Na+-High-Affinity Asp Binding Paradox in GltPh.

Glutamate transporters clear up excess extracellular glutamate by cotransporting three Na+ and one H+ with the countertransport of one K+. The archaeal homologs are selective to aspartate and only cotransport three Na+. The crystal structures of GltPh from archaea have been used in computational studies to understand the transport mechanism. Although some progress has been made with regard to the ligand-binding sites, a consistent picture of transport still eludes us. A major concern is the discrepancy between the computed binding free energies, which predict high-affinity Na+-low-affinity aspartate binding, and the experimental results in which the opposite is observed. Here, we show that the binding of the first two Na+ ions involves an intermediate state near the Na1 site, where two Na+ ions coexist and couple to aspartate with similar strengths, boosting its affinity. Binding free energies for Na+ and aspartate obtained using this intermediate state are in good agreement with the experimental values. Thus, the paradox in binding affinities arises from the assumption that the ligands bind to the sites observed in the crystal structure following the order dictated by their binding free energies with no intermediate states. In fact, the presence of an intermediate state eliminates such a correlation between the binding free energies and the binding order. The intermediate state also facilitates transition of the first Na+ ion to its final binding site via a knock-on mechanism, which induces substantial conformational changes in the protein consistent with experimental observations.

Insights into the transport side of the human SLC38A9 transceptor.

The lysosomal amino acid transporter SLC38A9 is referred to as transceptor, i.e. a transporter with a receptor function. The protein is responsible for coupling amino acid transport across the lysosomal membrane according to the substrate availability to mTORC1 signal transduction. This process allows cells to sense amino acid level responding to growth stimuli in physiological and pathological conditions triggering mTOR regulation. The main substrates underlying this function are glutamine and arginine. The functional and kinetic characterization of glutamine and arginine transport was performed using human SLC38A9 produced in E. coli, purified by affinity chromatography and reconstituted in liposomes. A cooperative behaviour for the wild type protein was revealed for both the substrates. A novel Na+ binding site, namely T453, was described by combined approaches of bioinformatics, site-directed mutagenesis and transport assay. Stimulation by cholesterol of glutamine and arginine transport was observed. The biological function of SLC38A9 relies on the interaction between its N-terminus and components of the mTOR complex; a deletion mutant of the N-terminus tail was produced and transport of glutamine was assayed revealing that this portion does not play any role in the intrinsic transport function of the human SLC38A9. Different features for glutamine and arginine transport were revealed: human SLC38A9 is competent for glutamine efflux, while that of arginine is negligible. In line with these results, imposed ∆pH stimulated glutamine, not arginine transport. Arginine plays, on the contrary, a modulatory function and is able to stimulate glutamine efflux. Interestingly, reciprocal inhibition experiments also supported by bioinformatics, suggested that glutamine and arginine may bind to different sites in the human SLC38A9 transporter.

Secondary Active Transporters.

Transport of solutes across biological membranes is essential for cellular life. This process is mediated by membrane transport proteins which move nutrients, waste products, certain drugs and ions into and out of cells. Secondary active transporters couple the transport of substrates against their concentration gradients with the transport of other solutes down their concentration gradients. The alternating access model of membrane transporters and the coupling mechanism of secondary active transporters are introduced in this book chapter. Structural studies have identified typical protein folds for transporters that we exemplify by the major facilitator superfamily (MFS) and LeuT folds. Finally, substrate binding and substrate translocation of the transporters LacY of the MFS and AdiC of the amino acid-polyamine-organocation (APC) superfamily are described.

Amino acid permeases in Cryptococcus neoformans are required for high temperature growth and virulence; and are regulated by Ras signaling.

Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.

Synthesis and evaluation of an 18F-labeled boramino acid analog of aminosuberic acid for PET imaging of the antiporter system xC.

In this study, we synthesized 18F-ASu-BF3, a close boramino acid analog of 5-[18F]fluoro-aminosuberic acid (18F-ASu), via 18F-19F isotope exchange reaction and evaluated its potential for imaging with positron emission tomography (PET). 18F-ASu-BF3 was stable in mouse plasma and taken up into PC3 prostate cancer cells via the system xC- amino acid transporter. The continuous use of isoflurane for anesthesia during dynamic imaging acquisition slowed down the excretion of 18F-ASu-BF3 and enabled visualization of PC3 tumor xenografts in mice. In contrast, no tumor visualization was observed from static images of 18F-BF3-ASu due to its rapid renal excretion mediated in part by the organic anion transporter. Our data indicate that the pharmacokinetics of amino acids could be altered after being converted into their boramino acid analogs. Therefore, care should be taken when using the boramino acid strategy to design and prepare 18F-labeled tracers for imaging amino acid transporters/receptors with PET.

Amino acid transporters revisited: New views in health and disease.

Amino acid transporters (AATs) are membrane-bound transport proteins that mediate transfer of amino acids into and out of cells or cellular organelles. AATs have diverse functional roles ranging from neurotransmission to acid-base balance, intracellular energy metabolism, and anabolic and catabolic reactions. In cancer cells and diabetes, dysregulation of AATs leads to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer, obesity and diabetes. Indeed, the neutral amino acid transporters (NATs) SLC7A5/LAT1 and SLC1A5/ASCT2 are likely involved in several human malignancies. However, a clinical therapy that directly targets AATs has not yet been developed. The purpose of this review is to highlight the structural and functional diversity of AATs, their diverse physiological roles in different tissues and organs, their wide-ranging implications in human diseases and the emerging strategies and tools that will be necessary to target AATs therapeutically.