Explore the vast range of titles available.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2. EAAT2 is an amino acid transporter implicated in glutamate homeostasis in brain and therapy resistance of cancer cells. Here, the authors report cryo-EM structures and reveal inhibitory mechanisms via selective inhibitor WAY213613.

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 Glt Ph and Glt Tk 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.

ObjectiveGlutamate transporters play a crucial role in neurotransmitter homeostasis, but studying their structure and function is challenging due to their membrane-bound nature. This study aims to investigate whether water-soluble QTY-variants of glutamate transporters EAA1, EAA2 and EAA3 retain the conformational characteristics and dynamics of native membrane-bound transporters.MethodsMolecular dynamics simulations and comparative genomics were used to analyze the structural dynamics of both native transporters and their QTY-variants. Native transporters were simulated in lipid bilayers, while QTY-variants were simulated in aqueous solution. Lipid distortions, relative solvent accessibilities, and conformational changes were examined. Evolutionary conservation profiles were correlated with structural dynamics. Statistical analyses included multivariate analysis to account for confounding variables.ResultsQTY-variants exhibited similar residue-wise conformational dynamics to their native counterparts, with correlation coefficients of 0.73 and 0.56 for EAA1 and EAA3, respectively (p < 0.001). Hydrophobic interactions of native helices correlated with water interactions of QTY- helices (rs = 0.4753, p < 0.001 for EAA1). QTY-variants underwent conformational changes resembling the outward-to-inward transition of native transporters.ConclusionsWater-soluble QTY-variants retain key structural properties of native glutamate transporters and mimic aspects of native lipid interactions, including conformational flexibility. This research provides valuable insights into the conformational changes and molecular mechanisms of glutamate transport, potentially offering a new approach for studying membrane protein dynamics and drug interactions.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b0,+AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b0,+AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

In-feed antibiotics have been commonly used to promote the growth performance of piglets. The antibiotics can increase protein utilization, but the underlying mechanism is largely unknown. The present study investigated the effects of in-feed antibiotics on intestinal AA transporters and receptors to test the hypothesis that the alteration of circulating AA profiles may be concomitant with the change of intestinal AA transporters and receptors. Sixteen litters of piglets at day 7 started to receive creep feed with (Antibiotic) or without (Control) antibiotic. Piglets were weaned at day 23 after birth, and fed the same diets until day 42. In-feed antibiotics did not affect the BW of 23-day-old ( P  = 0.248), or 42-day-old piglets ( P  = 0.089), but increased the weight gain to feed ratio from day 23 to 42 ( P  = 0.020). At day 42 after birth, antibiotic treatment increased the concentrations of most AAs in serum ( P  < 0.05), and decreased the concentrations of most AAs in jejunal and ileal digesta. Antibiotics upregulated ( P  < 0.05) the mRNA expression levels for jejunal AAs transporters (CAT1, EAAC1, ASCT2, y + LAT1), peptide transporters (PepT1), and Na + –K + –ATPase (ATP1A1), and ileal AA transporters (ASCT2, y + LAT1, b 0,+ AT, and B 0 AT1), and ATP1A1. The antibiotics also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR, and ileal T1R3. Protein expression levels for jejunal AA transporters (EAAC1, b 0,+ AT, and ASCT2) and PepT1 were also upregulated. Correlation analysis revealed that the alterations of AA profiles in serum after the in-feed antibiotics were correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. In conclusion, the in-feed antibiotics increased serum level of most AAs and decreased most AAs in the small intestine. These changes correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. The findings provide further insights into the mechanism of in-feed antibiotics, which may provide new framework for designing alternatives to antibiotics in animal feed in the future.

Epigenetic dysregulation, including accumulation of Histone H3 lysine 27 acetylation (H3K27ac), is a hallmark of pVHL-deficient clear cell Renal Cell Carcinomas (ccRCCs). Using an in vivo positive selection ORF screen in poorly tumorigenic pVHL-proficient cells and mechanistic studies in pVHL-deficient cells, we discovered that the aspartate (Asp) and glutamate (Glu) transporter, SLC1A1/EAAT3, is a metabolic dependency in ccRCC. pVHL loss promotes Hypoxia Inducible Factor (HIF)-independent SLC1A1 expression via H3K27ac dysregulation. SLC1A1 inactivation, genetically or pharmacologically, depletes Asp/Glu-derived metabolites (e.g., Tricarboxylic acid cycle and nucleotide intermediates), impedes ccRCC growth, and sensitizes ccRCCs to anti-metabolite drugs (e.g., glutaminase blockers). In human tumors, higher SLC1A1 expression is associated with reduced immune infiltration, oncogenic metabolic programs, and advanced stage/metastatic disease. Finally, in ccRCC animal models, SLC1A1 inactivation diminishes lung metastasis and the outgrowth of established renal tumors. Altogether, our studies credential SLC1A1 as an actionable, HIF-independent, metabolic dependency in pVHL-deficient ccRCCs. Clear cell renal cell carcinoma (ccRCC) bears the hallmark loss of VHL but remains incurable. Here, the authors identify the SLC1A1 dicarboxylic amino acid transporter as an actionable, oncogenic, HIF-independent, metabolic dependency in VHL-deficient ccRCCs.

Excitatory amino acid transporters (EAATs) are high-affinity Na⁺-dependent carriers of major importance in maintaining glutamate homeostasis in the central nervous system. EAAT3, the human counterpart of the rodent excitatory amino acid carrier 1 (EAAC1), is encoded by the SLC1A1 gene. EAAT3/EAAC1 is ubiquitously expressed in the brain, mostly in neurons but also in other cell types, such as oligodendrocyte precursors. While most of the glutamate released in the synapses is taken up by the “glial-type” EAATs, EAAT2 (GLT-1 in rodents) and EAAT1 (GLAST), the functional role of EAAT3/EAAC1 is related to the subtle regulation of glutamatergic transmission. Moreover, because it can also transport cysteine, EAAT3/EAAC1 is believed to be important for the synthesis of intracellular glutathione and subsequent protection from oxidative stress. In contrast to other EAATs, EAAT3/EAAC1 is mostly intracellular, and several mechanisms have been described for the rapid regulation of the membrane trafficking of the transporter. Moreover, the carrier interacts with several proteins, and this interaction modulates transport activity. Much less is known about the slow regulatory mechanisms acting on the expression of the transporter, although several recent reports have identified changes in EAAT3/EAAC1 protein level and activity related to modulation of its expression at the gene level. Moreover, EAAT3/EAAC1 expression is altered in pathological conditions, such as hypoxia/ischemia, multiple sclerosis, schizophrenia, and epilepsy. This review summarizes these results and provides an overall picture of changes in EAAT3/EAAC1 expression in health and disease.

Mutant isocitrate dehydrogenase 1 (mIDH1) drives tumorigenesis via producing oncometabolite R-2-hydroxyglutarate (R-2-HG) across various tumor types. However, mIDH1 inhibitors appear only effective in hematological tumors. The therapeutic benefit in solid tumors remains elusive, likely due to the complex tumor microenvironment. In this study, we discover that R-2-HG produced by IDH1-mutant tumor cells is preferentially imported into vascular endothelial cells and remodels mitochondrial respiration to promote tumor angiogenesis, conferring a therapeutic vulnerability in IDH1-mutant solid tumors. Mechanistically, SLC1A1, a Na + -dependent glutamate transporter that is preferentially expressed in endothelial cells, facilitates the influx of R-2-HG from the tumor microenvironment into the endothelial cells as well as the intracellular trafficking of R-2-HG from cytoplasm to mitochondria. R-2-HG hijacks SLC1A1 to promote mitochondrial Na + /Ca 2+ exchange, which activates the mitochondrial respiratory chain and fuels vascular endothelial cell migration in tumor angiogenesis. SLC1A1 deficiency in mice abolishes mIDH1-promoted tumor angiogenesis as well as the therapeutic benefit of mIDH1 inhibitor in solid tumors. Moreover, we report that HH2301, a newly discovered mIDH1 inhibitor, shows promising efficacy in treating IDH1-mutant cholangiocarcinoma in preclinical models. Together, we identify a new role of SLC1A1 as a gatekeeper of R-2-HG-mediated crosstalk between IDH1-mutant tumor cells and vascular endothelial cells, and demonstrate the therapeutic potential of mIDH1 inhibitors in treating IDH1-mutant solid tumors via disrupting R-2-HG-promoted tumor angiogenesis.

Obsessive-compulsive disorder (OCD) is a severe, chronic neuropsychiatric disorder with a strong genetic component. The SLC1A1 gene encoding the neuronal glutamate transporter EAAT3 has been proposed as a candidate gene for this disorder. Gene variants affecting SLC1A1 expression in human brain tissue have been associated with OCD. Several mouse models fully or partially lacking EAAT3 have shown no alterations in baseline anxiety-like or repetitive behaviors. We generated a transgenic mouse model (EAAT3glo) to achieve conditional, Cre-dependent EAAT3 overexpression and evaluated the overall impact of increased EAAT3 expression at behavioral and synaptic levels. Mice with EAAT3 overexpression driven by CaMKIIα-promoter (EAAT3glo/CMKII) displayed increased anxiety-like and repetitive behaviors that were both restored by chronic, but not acute, treatment with fluoxetine or clomipramine. EAAT3glo/CMKII mice also displayed greater spontaneous recovery of conditioned fear. Electrophysiological and biochemical analyses at corticostriatal synapses of EAAT3glo/CMKII mice revealed changes in NMDA receptor subunit composition and altered NMDA-dependent synaptic plasticity. By recapitulating relevant behavioral, neurophysiological, and psychopharmacological aspects, our results provide support for the glutamatergic hypothesis of OCD, particularly for the increased EAAT3 function, and provide a valuable animal model that may open novel therapeutic approaches to treat this devastating disorder.

Numerous basic studies have reported on the neuroprotective properties of several purine derivatives such as caffeine and uric acid (UA). Epidemiological studies have also shown the inverse association of appropriate caffeine intake or serum urate levels with neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson’s disease (PD). The well-established neuroprotective mechanisms of caffeine and UA involve adenosine A2A receptor antagonism and antioxidant activity, respectively. Our recent study found that another purine derivative, paraxanthine, has neuroprotective effects similar to those of caffeine and UA. These purine derivatives can promote neuronal cysteine uptake through excitatory amino acid carrier protein 1 (EAAC1) to increase neuronal glutathione (GSH) levels in the brain. This review summarizes the GSH-mediated neuroprotective effects of purine derivatives. Considering the fact that GSH depletion is a manifestation in the brains of AD and PD patients, administration of purine derivatives may be a new therapeutic approach to prevent or delay the onset of these neurodegenerative diseases.