Deficient Endoplasmic Reticulum-Mitochondrial Phosphatidylserine Transfer Causes Liver Disease.

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids.

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

Heteromeric Amino Acid Transporters in Brain: from Physiology to Pathology.

In humans, more than 50 transporters are responsible for the traffic and balance of amino acids within and between cells and tissues, and half of them have been associated with disease [1]. Covering all common amino acids, Heteromeric Amino acid Transporters (HATs) are one class of such transporters. This review first highlights structural and functional studies that solved the atomic structure of HATs and revealed molecular clues on substrate interaction. Moreover, this review focuses on HATs that have a role in the central nervous system (CNS) and that are related to neurological diseases, including: (i) LAT1/CD98hc and its role in the uptake of branched chain amino acids trough the blood brain barrier and autism. (ii) LAT2/CD98hc and its potential role in the transport of glutamine between plasma and cerebrospinal fluid. (iii) y+LAT2/CD98hc that is emerging as a key player in hepatic encephalopathy. xCT/CD98hc as a potential therapeutic target in glioblastoma, and (iv) Asc-1/CD98hc as a potential therapeutic target in pathologies with alterations in NMDA glutamate receptors.

GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.

Heteromeric amino acid transporters. In search of the molecular bases of transport cycle mechanisms.

Heteromeric amino acid transporters (HATs) are relevant targets for structural studies. On the one hand, HATs are involved in inherited and acquired human pathologies. On the other hand, these molecules are the only known examples of solute transporters composed of two subunits (heavy and light) linked by a disulfide bridge. Unfortunately, structural knowledge of HATs is scarce and limited to the atomic structure of the ectodomain of a heavy subunit (human 4F2hc-ED) and distant prokaryotic homologues of the light subunits that share a LeuT-fold. Recent data on human 4F2hc/LAT2 at nanometer resolution revealed 4F2hc-ED positioned on top of the external loops of the light subunit LAT2. Improved resolution of the structure of HATs, combined with conformational studies, is essential to establish the structural bases for light subunit recognition and to evaluate the functional relevance of heavy and light subunit interactions for the amino acid transport cycle.

Molecular basis of substrate-induced permeation by an amino acid antiporter.

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter.

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket.

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

Role of the transporter regulator protein (RS1) in the modulation of concentrative nucleoside transporters (CNTs) in epithelia.

SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na(+)-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.

Functional outcome of a novel SLC29A3 mutation identified in a patient with H syndrome.

The H syndrome (OMIM 612391) is an autosomal recessive disorder characterized by hyperpigmentation, hypertrichosis, histiocytosis and short stature. It is caused by mutations in the SLC29A3 gene, which encodes for the equilibrative nucleoside transporter 3 protein (ENT3), of still uncertain subcellular localisation. Here we report a new case of H syndrome with the novel mutation c.243delA, which has been concomitantly described by others [A. Bolze, A. Abhyankar, A.V. Grant, B. Patel, R. Yadav, M. Byun, D. Caillez, J.F. Emile, M. Pastor-Anglada, L. Abel, A. Puel, R. Govindarajan, L. de Pontual, J.L. Casanova, A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant, PLoS ONE 7 (2012) e29708]. Patient-derived primary skin fibroblasts and B-lymphoblastoid cell lines (B-LCL) were obtained and, although no differences were found in mRNA levels of ENT3, a significant increase in plasma membrane equilibrative transport activity was found in fibroblasts from the patient. Loss of function of key proteins implicated in nucleoside metabolism can lead to mitochondrial DNA (mtDNA) depletion syndromes (MDS). Measurement of respiratory chain complex activity revealed that mitochondrial function was unaltered. Neither fibroblasts nor B-LCL showed mtDNA depletion when compared with controls. Fibroblasts and B-LCL from the patient were not particularly protected when mitochondrial damage was induced using nucleoside-derived drugs susceptible to being transported by ENT3. Analysis of mtDNA amounts in tissues obtained at autopsy proved inconclusive with respect to mitochondrial involvement in the pathogenesis of this syndrome. Overall, the data do not support the inclusion of H syndrome among the MDS and these findings are compatible with its recent inclusion among the lysosomal storage diseases.

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.

Expression and distribution of nucleoside transporter proteins in the human syncytiotrophoblast.

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.

Membrane Protein Stabilization Strategies for Structural and Functional Studies.

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

Rush Hour of LATs towards Their Transport Cycle.

The mammalian SLC7 family comprises the L-amino acid transporters (LATs) and the cationic amino acid transporters (CATs). The relevance of these transporters is highlighted by their involvement in several human pathologies, including inherited rare diseases and acquired diseases, such as cancer. In the last four years, several crystal or cryo-EM structures of LATs and CATs have been solved. These structures have started to fill our knowledge gap that previously was based on the structural biology of remote homologs of the amino acid-polyamine-organocation (APC) transporters. This review recovers this structural and functional information to start generating the molecular bases of the transport cycle of LATs. Special attention is given to the known transporter conformations within the transport cycle and the molecular bases for substrate interaction and translocation, including the asymmetric interaction of substrates at both sides of the plasma membrane.

Different N-terminal motifs determine plasma membrane targeting of the human concentrative nucleoside transporter 3 in polarized and nonpolarized cells.

Human concentrative nucleoside transporter 3 (hCNT3) is a broad-selectivity, high-affinity protein implicated in the uptake of most nucleoside-derived anticancer and antiviral drugs. Regulated trafficking of hCNT3 has been recently postulated as a suitable way to improve nucleoside-based therapies. Moreover, the recent identification of a putative novel hCNT3-type transporter lacking the first 69 amino acids and retained at the endoplasmic reticulum anticipated that the N terminus of hCNT3 contains critical motifs implicated in trafficking. In the current study, we have addressed this issue by using deletions and site-directed mutagenesis and plasma membrane expression and nucleoside uptake kinetic analysis. Data reveal that 1) a segment between amino acids 50 and 62 contains plasma membrane-sorting determinants in nonpolarized cells; 2) in particular, the Val(57)-Thr(58)-Val(59) tripeptide seems to be the core of the export signal, whereas acidic motifs upstream and downstream of it seem to be important for the kinetics of the process; and 3) in polarized epithelia, the ß-turn-forming motif (17)VGFQ(20) is necessary for proper apical expression of the protein.

The human concentrative nucleoside transporter-3 C602R variant shows impaired sorting to lipid rafts and altered specificity for nucleoside-derived drugs.

The human concentrative nucleoside transporter-3 C602R (hCNT3C602R), a recently identified human concentrative nucleoside transporter-3 (hCNT3) variant, has been shown to interact with natural nucleosides with apparent K(m) values similar to those of the wild-type transporter, although binding of one of the two sodium ions required for nucleoside translocation is impaired, resulting in decreased V(max) values (Mol Pharmacol 73:379-386, 2008). We have further analyzed the properties of this hCNT3 variant by determining its localization in plasma membrane lipid domains and its interaction with nucleoside-derived drugs used in anticancer and antiviral therapies. When expressed heterologously in HeLa cells, wild-type hCNT3 localized to both lipid raft and nonlipid raft domains. Treatment of cells with the cholesterol-depleting agent methyl-beta-cyclodextrin resulted in a marked decrease in hCNT3-related transport activity that was associated with the loss of wild-type hCNT3 from lipid rafts. It is noteworthy that although exogenously expressed hCNT3C602R was present in nonlipid raft domains at a level similar to that of the wild-type transporter, the mutant transporter was present at much lower amounts in lipid rafts. A substrate profile analysis showed that interactions with a variety of nucleoside-derived drugs were altered in the hCNT3C602R variant and revealed that sugar hydroxyl residues are key structural determinants for substrate recognition by the hCNT3C602R variant.

A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum.

Nucleoside transporters are plasma membrane proteins essential for nucleoside salvage. Among them, human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) plays an essential role in this process because of its broader substrate selectivity and higher concentrative ability than the other members of the SLC28 protein family, hCNT1 and hCNT2. The aim of this study was to characterize an isoform of hCNT3, encoded by an alternatively spliced SLC28A3-related mRNA, the first identified for a CNT protein. This variant, named hCNT3ins, is the result of the insertion of 176 bp corresponding to an intron located between exons 2 and 3 of the gene. This insertion results in a shift of the reading frame, yielding a protein lacking 69 residues of the N terminus. hCNT3 and hCNT3ins mRNAs are simultaneously expressed both in normal and transformed cells and are differentially regulated by activation and differentiation. Because of the N-terminal deletion, hCNT3ins is retained in the endoplasmic reticulum, where it shows a typical hCNT3-related activity. hCNT3ins exhibits a shorter half-life than its plasma membrane counterpart, being degraded via a proteasome-dependent pathway. We suggest that this novel hCNT3 isoform would be involved in the salvage of intracellular nucleosides from the lumen of the endoplasmic reticulum to the cytoplasm.

Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7.

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites-a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.