Identification of LAT4, a novel amino acid transporter with system L activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt

Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease

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

Incorporating the sex and gender perspective in research content: a toolkit

Perspectiva de gènere; Recerca de la salutPerspectiva de género; Investigación de la saludGender perspective; Health researchAquest document, d'una banda, argumenta mitjançant exemples concrets, perquè és important considerar el sexe i el gènere en la recerca de la salut. I de l'altra, presenta una eina per integrar la perspectiva de gènere i sexe per guiar el personal investigador a l'hora de considerar la incorporació del sexe o el gènere en estudis de ciència bàsica, clínica, de serveis sanitaris i de salut pública. L'eina està formada per una llista de preguntes que aborden la integració del sexe/gènere a la recerca, una llista de subpreguntes que permeten il·lustrar cada pregunta amb aspectes concrets i unes aportacions extres amb exemples sobre com incorporar els aspectes de sexe i gènere. L'eina recull cada fase del procés de recerca

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Caracterització de LAT4 i EEG1, dos membres de la família de transportadors d'aminoàcids SLC43

[cat] Els transportadors del sistema L faciliten el moviment d'aminoàcids grans i neutres a través de les membranes cel·lulars. Fins el moment es coneixien 3 proteïnes capaces d'induir aquesta activitat de transport: LAT1, LAT2 i LAT3. En aquets treball s'ha identificat LAT4, un nou transportador d'aminoàcids de la família SLC43 que guarda un 57% i un 30% d'identitat amb LAT3 i EEG1, els altres dos membres de la família. La proteïna es glicosila i arriba a la membrana plasmàtica de les cèl·lules HeLa i dels oòcits de Xenopus laevis quan es sobreexpressa. El RNA missatger de LAT4 s'expressa majoritàriament a placenta, intestí i ronyó. Més concretament, mitjançant hibridació in situ, hem detectat el missatger de LAT4 a cèl·lules dels túbuls distals i dels conductes col·lectors del ronyó, i a les cèl·lules de la cripta i la base de l'enteròcit a l'intestí prim. L'expressió de LAT4 en oòcits de Xenopus laevis indueix una activitat de transport d'aminoàcids neutres i grans; sodi, clorur i pH independent i que s'inhibeix per l'anàleg d'aminoàcids no metabolitzable BCH. Aquestes propietats concorden amb el subsistema prèviament descrit L2 i afegeixen un quart transportador responsable de l'activitat del sistema L. L'activitat de transport induïda, no és transestimulable, i possiblement es tracti d'un mecanisme de difusió facilitada. El transport induït per LAT4 mostra una cinètica de dos components, on les constants d'afinitat són de 4,7+/-0,5 mM i 178+/-29 mil.limicres pel component de baixa i alta afinitat, respectivament. L'agent alquilant de grups sulfidrils, N-etilmaleïmida (NEM) inhibeix parcialment l'activitat de transport induïda per LAT4 i efecte específicament el component de baixa afinitat. A més a més, hem identificat el mutant LAT4 S297A insensible al pretractament amb NEM. Al model cel·lular de ronyó PCT, es detecta una activitat de transport de baixa afinitat a la cara basolateral compatible amb la descrita per LAT4. EEG1, el tercer membre de la família SLC43, es glicosila i expressat en sistemes heteròlegs arriba a la membrana plasmàtica de la cèl·lula, però no indueix activitat de transport pels aminoàcids assajats. El RNA missatger de EEG1 es detecta majoritàriament a cor, fetge, i en menor grau a ronyó, pulmó i placenta, tant en teixits humans com de ratolí. S'ha generat un anticòs contra la proteïna que permet la seva detecció a ronyó, intestí i pulmó amb diferents patrons de glicosilació. També s'ha generat un model murí amb EEG1 mutat, mitjançant l'exposició a l'agent altament mutagènic etilnitrosurea (ENU). La mutació puntual detectada, introdueix un codó stop prematur concretament a la posició de la tirosina 221, que trunca la proteïna aproximadament a la meitat. Els ratolins homozigots per la mutació són viables, fèrtils i segueixen una herència mendeliana. No existeixen diferències pel que fa al pes dels animals mutants i control. No hiperexcreten aminoàcids en orina, ni tenen nivells elevats d'aminoàcids en sang. Presenten alteracions histopatològiques al ronyó i al fetge. Suggerim que EEG1 no té un paper rellevant en la reabsorció renal d'aminoàcids sense descartar una possible compensació per altres transportadors.[eng] System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now, three proteins that induce system L activity have been identified: LAT1, LAT2 and LAT3. In the present study we identify a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3 and 30% to EEG1. LAT4 protein is glycosylated and reaches plasma membrane in HeLa cells and Xenopus oocytes when LAT4 is overexpressed. LAT4 mRNA is expressed mainly in placenta, small intestine and kidney. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of distal tubule and the collecting duct in the kidney and in the cells of the crypt in the intestine. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide (NEM) but not that with high affinity. Mutation in LAT4 of the SLC43 conserved S 297 to A abolishes sensitivity to NEM. EEG1, the third member of SLC43 is glycosylated and when is expressed in heterologous system it reaches the plasma membrane. EEG1 mRNA is expressed mainly in heart and liver, but also is present in kidney, lung and placenta. We generated an antibody able to recognize EEG1 from kidney, small intestine and lung with different patterns of glycosylation. We also generated an EEG1 mutated mouse model caused by the chemical mutagen N-ethyl-N-nitrosurea (ENU) which introduced a point mutation resulting in a truncated protein in amino acid position 221. The homozygous mutant mice were born at normal Mendelian ratios when intercrossing between the heterozygous mice, indicating that there was no embryonic mortality in the mutant animals. Homozygous mice do not hyperexcrete any amino acid in urine and no high level of amino acids in plasma were found. Histopathological studies of the kidney and liver reveal damage alterations in both tissues

The small SLC43 family: facilitator system l amino acid transporters and the orphan EEG1

The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein

Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids

Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter yLAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by studies but has not been evaluated To study the functional relationship of TAT1 and LAT2/CD98hc , we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of yLAT1/CD98hc. The LAT2/CD98hc and TAT1 transporters functionally cooperate , and yLAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans

La ablación del transportador de aminoácidos LAT2 (SLC7A8) causa hipoacusia asociada al envejecimiento en ratón

Trabajo presentado a la X Reunión Científica Anual CIBERER, celebrada en San Lorenzo del Escorial del 23 al 24 de marzo de 2017.Peer Reviewe

Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in Age-Related Hearing Loss.

Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations