RESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Cadena Pesada de la Proteína-1 Reguladora de Fusión , Humanos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/química , Ligandos , Simulación de Dinámica MolecularRESUMEN
Sarcopenia is a major contributor to disability in older adults, and thus, it is key to elucidate the mechanisms underlying its development. Increasing evidence suggests that impaired macroautophagy/autophagy contributes to the development of sarcopenia. However, the mechanisms leading to reduced autophagy during aging remain largely unexplored, and whether autophagy activation protects from sarcopenia has not been fully addressed. Here we show that the autophagy regulator TP53INP2/TRP53INP2 is decreased during aging in mouse and human skeletal muscle. Importantly, chronic activation of autophagy by muscle-specific overexpression of TRP53INP2 prevents sarcopenia and the decline of muscle function in mice. Acute re-expression of TRP53INP2 in aged mice also improves muscle atrophy, enhances mitophagy, and reduces ROS production. In humans, high levels of TP53INP2 in muscle are associated with increased muscle strength and healthy aging. Our findings highlight the relevance of an active muscle autophagy in the maintenance of muscle mass and prevention of sarcopenia.Abbreviation: ATG7: autophagy related 7; BMI: body mass index; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ROS: reactive oxygen species; TP53INP2: tumor protein p53 inducible nuclear protein 2; WT: wild type.
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Autofagia , Músculo Esquelético , Sarcopenia , Animales , Humanos , Masculino , Ratones , Autofagia/fisiología , Envejecimiento Saludable/fisiología , Envejecimiento Saludable/metabolismo , Ratones Endogámicos C57BL , Mitofagia/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Proteínas Nucleares , Especies Reactivas de Oxígeno/metabolismo , Sarcopenia/patología , Sarcopenia/metabolismoRESUMEN
In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities.
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Calcio , Retículo Endoplásmico , GTP Fosfohidrolasas , Mitocondrias , Proteínas Mitocondriales , Animales , Ratones , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Hígado/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Isoformas de Proteínas , Ratones Noqueados , Humanos , Ratones Endogámicos C57BL , Células HeLa , Empalme Alternativo , Estrés del Retículo EndoplásmicoRESUMEN
Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.
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ADN Mitocondrial , Miositis , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Receptor Toll-Like 9/metabolismo , Dinámicas Mitocondriales/genética , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/patología , Inflamación/patologíaRESUMEN
Brown adipose tissue (BAT) thermogenesis affects energy balance, and thereby it has the potential to induce weight loss and to prevent obesity. Here, we document a macroautophagic/autophagic-dependent mechanism of peroxisome proliferator-activated receptor gamma (PPARG) activity regulation that induces brown adipose differentiation and thermogenesis and that is mediated by TP53INP2. Disruption of TP53INP2-dependent autophagy reduced brown adipogenesis in cultured cells. In vivo specific-tp53inp2 ablation in brown precursor cells or in adult mice decreased the expression of thermogenic and mature adipocyte genes in BAT. As a result, TP53INP2-deficient mice had reduced UCP1 content in BAT and impaired maximal thermogenic capacity, leading to lipid accumulation and to positive energy balance. Mechanistically, TP53INP2 stimulates PPARG activity and adipogenesis in brown adipose cells by promoting the autophagic degradation of NCOR1, a PPARG co-repressor. Moreover, the modulation of TP53INP2 expression in BAT and in human brown adipocytes suggests that this protein increases PPARG activity during metabolic activation of brown fat. In all, we have identified a novel molecular explanation for the contribution of autophagy to BAT energy metabolism that could facilitate the design of therapeutic strategies against obesity and its metabolic complications.
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Tejido Adiposo Pardo , PPAR gamma , Ratones , Humanos , Animales , Tejido Adiposo Pardo/metabolismo , PPAR gamma/metabolismo , Autofagia , Obesidad/metabolismo , Termogénesis/genética , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismoRESUMEN
How the complex environment of a membrane protein interplays with its structure and function is illustrated in the review by Jimenez-Munguia on interferon-induced transmembrane protein 3 (IFITM3), an antiviral protein that blocks fusion of the viral membrane with the host cell. In their review, they touch upon the possibility of IFITM3 adopting different topologies which overlays with the dependence of its activity on the local lipid composition of the membrane, thus making for an intricate mechanistic question which is yet to be understood in complete molecular detail.
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Antivirales , Proteínas de la Membrana , Proteínas de la Membrana/químicaRESUMEN
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.
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Sistemas de Transporte de Aminoácidos , Biología Molecular , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico/fisiología , Humanos , Especificidad por SustratoRESUMEN
Sarcopenia is one of the main factors contributing to the disability of aged people. Among the possible molecular determinants of sarcopenia, increasing evidences suggest that chronic inflammation contributes to its development. However, a key unresolved question is the nature of the factors that drive inflammation during aging and that participate in the development of sarcopenia. In this regard, mitochondrial dysfunction and alterations in mitophagy induce inflammatory responses in a wide range of cells and tissues. However, whether accumulation of damaged mitochondria (MIT) in muscle could trigger inflammation in the context of aging is still unknown. Here, we demonstrate that BCL2 interacting protein 3 (BNIP3) plays a key role in the control of mitochondrial and lysosomal homeostasis, and mitigates muscle inflammation and atrophy during aging. We show that muscle BNIP3 expression increases during aging in mice and in some humans. BNIP3 deficiency alters mitochondrial function, decreases mitophagic flux and, surprisingly, induces lysosomal dysfunction, leading to an upregulation of Toll-like receptor 9 (TLR9)-dependent inflammation and activation of the NLRP3 (nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3) inflammasome in muscle cells and mouse muscle. Importantly, downregulation of muscle BNIP3 in aged mice exacerbates inflammation and muscle atrophy, and high BNIP3 expression in aged human subjects associates with a low inflammatory profile, suggesting a protective role for BNIP3 against age-induced muscle inflammation in mice and humans. Taken together, our data allow us to propose a new adaptive mechanism involving the mitophagy protein BNIP3, which links mitochondrial and lysosomal homeostasis with inflammation and is key to maintaining muscle health during aging.
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Sarcopenia , Envejecimiento , Animales , Homeostasis , Humanos , Inflamación/metabolismo , Lisosomas/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Atrofia Muscular/metabolismo , Sarcopenia/metabolismoRESUMEN
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.
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Sistemas de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Transporte Biológico , Barrera Hematoencefálica/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/fisiología , Especificidad por Sustrato/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/fisiología , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Aminoácidos Neutros/metabolismo , Transporte Biológico/fisiología , Microscopía por Crioelectrón/métodos , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Células HeLa , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , alfa-Glucosidasas/química , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Dominio Catalítico , Microscopía por Crioelectrón , Cadena Pesada de la Proteína-1 Reguladora de Fusión/química , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Humanos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína , alfa-Glucosidasas/genéticaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Aminoácidos/metabolismo , Metabolismo Energético , Homeostasis , Inflamación/etiología , Inflamación/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Transporte Biológico , Biomarcadores , Susceptibilidad a Enfermedades , HumanosRESUMEN
BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/líquido cefalorraquídeo , Células Epiteliales/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Homeostasis , Sistema de Transporte de Aminoácidos y+/genética , Animales , Transporte Biológico/genética , Transporte Biológico/fisiología , Células Cultivadas , Plexo Coroideo/metabolismo , Femenino , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/genética , Ácido Glutámico/metabolismo , Homeostasis/fisiología , Masculino , Ratones Noqueados , Prealbúmina/metabolismoRESUMEN
CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.
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Aminoácidos/metabolismo , Ciclo Celular , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Nucleótidos/metabolismo , Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , División Celular , Daño del ADN , Reparación del ADN , Cadena Pesada de la Proteína-1 Reguladora de Fusión/fisiología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Estrés OxidativoRESUMEN
Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y+LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7-/-). The Slc7a7-/- model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7-/- mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7-/- model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression.
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Errores Innatos del Metabolismo de los Aminoácidos/patología , Sistema de Transporte de Aminoácidos y+L/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Sistema de Transporte de Aminoácidos y+L/genética , Aminoácidos/metabolismo , Animales , Citrulina/uso terapéutico , Modelos Animales de Enfermedad , Hiperamonemia/metabolismo , Hiperamonemia/patología , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteinosis Alveolar Pulmonar/metabolismo , Proteinosis Alveolar Pulmonar/patologíaRESUMEN
Cataract, the loss of ocular lens transparency, accounts for â¼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects.
RESUMEN
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.
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GTP Fosfohidrolasas/metabolismo , Proteínas Mitocondriales/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosfatidilserinas/metabolismo , Animales , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Inflamación/metabolismo , Hígado/patología , Hepatopatías/etiología , Hepatopatías/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Cultivo Primario de Células , Transporte de Proteínas/fisiología , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
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.