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1.
J Anim Sci ; 98(2)2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958134

RESUMO

Vitamin B6 (VB6), which is an essential functional substance for biosome, plays an irreplaceable role in animal health. However, there are few studies that focus on the correlation between VB6 and intestinal health in weaned piglets. This study was conducted to investigate the effects of VB6 on the growth performance, intestinal morphology, and inflammatory cytokines and amino acid (AA) transporters mRNA expression in weaned piglets that are fed a low crude-protein (CP, 18%) diet. Eighteen crossbred piglets with initial body weights of 7.03 ± 0.17 kg (means ± SEM), weaned at 21-d age, were randomly assigned three diets with 0, 4, and 7 mg/kg VB6 supplementation, respectively. The experimental period lasted 14 days. Our results showed that there were no significant differences in growth performance, diarrhea rate, and biochemical parameters among the three treatments. In the jejunum, dietary VB6 supplementation did not affect the morphology and positive Ki67 counts. Dietary supplementation with 4 mg/kg VB6 decreased the mRNA expression of COX-2, IL-10, and TGF-ß (P < 0.05). Dietary supplementation with 7 mg/kg VB6 increased the mRNA expression of SLC7A1, SLC7A6, SLC16A14, and SLC38A5 (P < 0.05) and 4 or 7 mg/kg VB6 decreased SLC36A1 mRNA expression (P < 0.05). In the ileum, VB6 supplementation did not affect positive Ki67 counts but significantly decreased villus area (P < 0.05) and tended to decrease villus height (P = 0.093). Dietary supplementation with 4 mg/kg VB6 had significantly increased the mRNA expression of IL-1ß, TNF-α, COX-2, IL-10, and TGF-ß (P < 0.05). Dietary supplementation with 4 or 7 mg/kg VB6 had significantly decreased SLC6A20, SLC7A1, SLC7A6, SLC16A14, and SLC38A5 mRNA expression (P < 0.05). These findings suggest that dietary supplementation of VB6 mainly down-regulated inflammatory cytokines and up-regulated AA transporters mRNA expression in jejunum, while up-regulated (4 mg/kg) inflammatory cytokines and down-regulated AA transporters mRNA expression in ileum, which may provide a reference for the intestinal development of weaned piglets that are fed a low-CP diet.


Assuntos
Dieta com Restrição de Proteínas/veterinária , Suplementos Nutricionais/análise , Suínos/fisiologia , Vitamina B 6/administração & dosagem , Sistemas de Transporte de Aminoácidos/metabolismo , Ração Animal/análise , Animais , Biomarcadores/sangue , Citocinas/metabolismo , Diarreia/veterinária , Dieta/veterinária , Regulação da Expressão Gênica , Inflamação/veterinária , Intestinos/crescimento & desenvolvimento , Intestinos/fisiologia , Distribuição Aleatória , Suínos/crescimento & desenvolvimento , Desmame
2.
J Dairy Sci ; 103(3): 2847-2863, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31928756

RESUMO

Branched-chain amino acids (BCAA) are major components of milk protein and important precursors for nonessential AA. Thus, the BCAA transport and break-down play a key role in the metabolic adaptation to the high nutrient demands in lactation. However, in monogastrics, increased BCAA levels have been linked with obesity and certain metabolic disorders such as impaired insulin sensitivity. Our objective was to study the effect of over-conditioning at calving on plasma BCAA levels as well as the tissue abundance of the most relevant BCAA transporters and degrading enzymes in dairy cows during late pregnancy and early lactation. Thirty-eight Holstein cows were allocated 15 wk antepartum to either a normal- (NBCS) or over-conditioned (HBCS) group, receiving 6.8 or 7.2 MJ of NEL/kg of DM, respectively, during late lactation to reach the targeted differences in body condition score (BCS) and back fat thickness (BFT; NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until dry-off. During the dry period and next lactation, cows were fed the same diets, whereby differences in BCS and BFT were maintained: prepartum means were 3.16 ± 0.06 and 1.03 ± 0.07 cm (NBCS) vs. 3.77 ± 0.08 and 1.89 ± 0.11 cm (HBCS), postpartum means were 2.89 ± 0.06 and 0.81 ± 0.05 cm (NBCS) vs. 3.30 ± 0.06 and 1.38 ± 0.08 cm (HBCS). Blood and biopsies from liver, semitendinosus muscle, and subcutaneous adipose tissue (scAT) were sampled at d 49 antepartum, 3, 21, and 84 postpartum. Free BCAA were analyzed and the mRNA abundance of solute carrier family 1 member 5 (SLC1A5), SLC7A5, and SLC38A2 as well as branched-chain aminotransferase 2 (BCAT2), branched-chain α-keto acid dehydrogenase E1α (BCKDHA), and branched-chain α-keto acid dehydrogenase E1ß (BCKDHB) as well as the protein abundance of BCKDHA were assessed. Concentrations of all BCAA changed with time, most markedly in HBCS cows, with a nadir around calving. Apart from Ile, neither individual nor total BCAA differed between groups. The HBCS group had greater BCKDHA mRNA as well as higher prepartum BCKDHA protein abundance in scAT than NBCS cows, pointing to a greater oxidative capacity for the irreversible degradation of BCAA transamination products in scAT of over-conditioned cows. Prepartum hepatic BCKDHA protein abundance was lower in HBCS than in NBCS cows. In both groups, SLC1A5, SLC7A5, and BCAT2 mRNA were most abundant in scAT, whereas SLC38A2 was higher in scAT and muscle compared with liver, and BCKDHA and BCKDHB mRNA were greatest in liver and muscle, respectively. Our results indicate that scAT may be a major site of BCAA uptake and initial catabolism, with the former, however, being independent of BCS and time relative to calving in dairy cows.


Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Bovinos/fisiologia , Leite/química , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/sangue , Animais , Bovinos/genética , Dieta/veterinária , Feminino , Lactação , Fígado/metabolismo , Músculo Esquelético/enzimologia , Período Pós-Parto , Gravidez , RNA Mensageiro/genética , Gordura Subcutânea/enzimologia
3.
Am J Physiol Gastrointest Liver Physiol ; 318(1): G189-G202, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31760764

RESUMO

The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Colo/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Animais , Bactérias/metabolismo , Colo/microbiologia , Fezes/química , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Cinética , Ratos Sprague-Dawley , Transcriptoma
4.
J Sci Food Agric ; 100(4): 1718-1725, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31821574

RESUMO

BACKGROUND: l-Theanine has multiple beneficial biological activities. However, there is little information about the use of l-theanine in broiler production. Therefore, this study investigated the effect of l-theanine on growth performance, intestinal development and health, and the mRNA levels of intestinal peptide and amino acid (AA) transporters of broilers. RESULTS: Body weight and average daily gain were increased by l-theanine, whereas feed to gain ratio was decreased (quadratic, P < 0.05). Notably, the relative weight of duodenum, jejunum and ileum, villus height, villus height to crypt depth ratio, the jejunal activities of glutathione peroxidase, total antioxidant capacity, catalase and total superoxide dismutase were increased linearly and/or quadratically by l-theanine (P < 0.05), whereas crypt depth, serum d-lactic acid, and jejunal protein carbonyls and malondialdehyde content were decreased linearly and/or quadratically (P < 0.05). Moreover, l-theanine enhanced the jejunal mRNA levels of occludin, claudin-1, E-cadherin, zona occludens-1, di- and tripeptide transporter, excitatory AA transporter 3, Na+ -independent cationic AA transporter 1, Na+ -independent cationic and zwitterionic AA transporter, Na+ - and Cl- -dependent neutral and cationic AA transporter, Na+ -independent cationic and Na+ -dependent neutral AA transporter (y+LAT) 1, y+LAT2, Na+ -independent branched-chain and aromatic AA transporter, and heavy chain corresponding to the b°,+ transport system (linear and/or quadratic, P < 0.05). CONCLUSIONS: l-Theanine beneficially affected the growth performance of broilers by improving intestinal development and health, and the intestinal mRNA levels of AA and peptide transporters. Therefore, l-theanine has the potential to be a promising feed additive for broilers. © 2020 Society of Chemical Industry.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Glutamatos/metabolismo , Intestinos/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Ração Animal/análise , Animais , Catalase/genética , Catalase/metabolismo , Galinhas/genética , Suplementos Nutricionais/análise , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
5.
J Sci Food Agric ; 100(1): 235-244, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31512251

RESUMO

BACKGROUND: This study evaluated the effects of early antibiotic exposure (EAE) on subsequent amino acid (AA) profiles and small intestinal AA transporter and receptor expression level in pigs with different dietary crude protein (CP) levels. Eighteen litters of piglets were fed creep feed diets, either with or without antibiotics while with sow on day 7. The pigs were weaned at day 23 and fed the same diets until day 42, when random pigs within each group were offered a normal- or low-CP diet, thereby creating four groups. On day 120, the pigs were euthanized, and jejunal and ileal mucosa and digesta were collected for gene-expression and AA-concentration analysis. RESULTS: With the normal-CP diet, EAE increased (P < 0.05) the concentrations of six essential amino acids (EAA) and three non-essential amino acids (NEAA) in serum, four EAAs and four NEAAs in jejunal mucosa, one EAA and two NEAAs in ileal mucosa, five EAAs and three NEAAs in jejunal digesta, and three EAAs and two NEAAs in ileal digesta. Early antibiotic exposure upregulated (P < 0.05) CAT1, ASCT2, ATB0,+ , CaSR, T1R1, and T1R3 expression in the jejunum, downregulated PepT1 expression with a normal-CP diet. It upregulated (P < 0.05) the expressions of CAT1, ATB0,+ , ATP1A1, and T1R3 in the ileum with a normal-CP diet. CONCLUSION: These results suggest that EAE has long-term effects on AA profiles, mainly in the jejunum and serum, by increasing AA transporter expression in the intestine, and that these effects may be influenced by dietary CP levels. © 2019 Society of Chemical Industry.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/metabolismo , Antibacterianos/efeitos adversos , Mucosa Intestinal/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/genética , Suínos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/química , Ração Animal/análise , Animais , Antibacterianos/administração & dosagem , Proteínas na Dieta/análise , Proteínas na Dieta/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Masculino , Distribuição Aleatória , Receptores Acoplados a Proteínas-G/metabolismo , Suínos/genética , Suínos/crescimento & desenvolvimento , Fatores de Tempo
6.
J Exp Med ; 217(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31649036

RESUMO

Foxp3+ regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance.


Assuntos
Tolerância Imunológica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Nutrientes/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Sistemas de Transporte de Aminoácidos/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Serina-Treonina Quinases TOR/imunologia
7.
Int J Mol Sci ; 20(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842320

RESUMO

The solute carrier (SLC) family-38 of transporters has eleven members known to transport amino acids, with glutamine being a common substrate for ten of them, with SLC38A9 being the exception. In this study, we examine the subcellular localization of SNAT10 in several independent immortalized cell lines and stem cell-derived neurons. Co-localization studies confirmed the SNAT10 was specifically localized to secretory organelles. SNAT10 is expressed in both excitatory and inhibitory neurons in the mouse brain, predominantly in the endoplasmic reticulum, and in the Golgi apparatus. Knock-down experiments of SNAT10, using Slc38a10-specific siRNA in PC12 cells reduced nascent protein synthesis by more than 40%, suggesting that SNAT10 might play a role in signaling pathways that regulate protein synthesis, and may act as a transceptor in a similar fashion to what has been shown previously for SLC38A2 (SNAT2) and SNAT9(SLC38A9).


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Biossíntese de Proteínas , Sistemas de Transporte de Aminoácidos/genética , Animais , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/metabolismo , Camundongos , Neurônios/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , Ratos
8.
Int J Mol Sci ; 21(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31878022

RESUMO

Amino acid transporters play very important roles in nutrient uptake, neurotransmitter recycling, protein synthesis, gene expression, cell redox balance, cell signaling, and regulation of cell volume. With regard to transporters that are closely connected to metabolism, amino acid transporter-associated diseases are linked to metabolic disorders, particularly when they involve different organs, cell types, or cell compartments. To date, 65 different human solute carrier (SLC) families and more than 400 transporter genes have been identified, including 11 that are known to include amino acid transporters. This review intends to summarize and update all the conditions in which a strong association has been found between an amino acid transporter and an inherited metabolic disorder. Many of these inherited disorders have been identified in recent years. In this work, the physiological functions of amino acid transporters will be described by the inherited diseases that arise from transporter impairment. The pathogenesis, clinical phenotype, laboratory findings, diagnosis, genetics, and treatment of these disorders are also briefly described. Appropriate clinical and diagnostic characterization of the underlying molecular defect may give patients the opportunity to avail themselves of appropriate therapeutic options in the future.


Assuntos
Sistemas de Transporte de Aminoácidos , Aminoácidos , Erros Inatos do Metabolismo , Transdução de Sinais/genética , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo/genética , Humanos , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia
9.
Int J Mol Sci ; 20(23)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31766598

RESUMO

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits.


Assuntos
Sistemas de Transporte de Aminoácidos/biossíntese , Citrullus/metabolismo , Frutas/metabolismo , Proteínas de Plantas/biossíntese , Sistemas de Transporte de Aminoácidos/genética , Arginina/genética , Arginina/metabolismo , Citrulina/genética , Citrulina/metabolismo , Citrullus/genética , Frutas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
10.
Nat Commun ; 10(1): 4851, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649258

RESUMO

Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteostase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Redobramento de Proteína
11.
Proc Natl Acad Sci U S A ; 116(42): 21047-21053, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570606

RESUMO

The placenta is critical in mammalian embryonic development because the embryo's supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through it. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on system A amino acid transporters Slc38a1/SNAT1, Slc38a2/SNAT2, and Slc38a4/SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4/SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4/SNAT4 in the placenta, paternal knockout (KO) but not maternal KO of Slc38a4/SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal-fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4/SNAT4 mutant embryos. These results indicated that SNAT4-mediated amino acid transport in mice plays a major role in placental and embryonic development. Given that expression of Slc38a4 in the placenta is conserved in other species, our Slc38a4/SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Placenta/metabolismo , Placenta/patologia , Útero/metabolismo , Útero/patologia , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Animais , Feminino , Camundongos , Placentação/fisiologia , Gravidez , Trofoblastos/metabolismo , Trofoblastos/patologia
12.
Biotechnol Lett ; 41(12): 1423-1431, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31650421

RESUMO

OBJECTIVE: The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation. RESULTS: Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells. CONCLUSIONS: Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.


Assuntos
Aminoácidos/metabolismo , Autofagia , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Pichia/crescimento & desenvolvimento , Pichia/genética , Deleção de Sequência , Sistemas de Transporte de Aminoácidos/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
13.
Sci Adv ; 5(9): eaax6352, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31555743

RESUMO

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome-associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.


Assuntos
Sistemas de Transporte de Aminoácidos , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Resistencia a Medicamentos Antineoplásicos , Melanoma Experimental , Proteínas de Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Simportadores , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/genética , Simportadores/genética , Simportadores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Nature ; 572(7771): 614-619, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31435015

RESUMO

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.


Assuntos
Tecido Adiposo Marrom/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Metabolismo Energético , Homeostase , Proteínas Mitocondriais/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Termogênese , Tecido Adiposo Marrom/citologia , Animais , Temperatura Baixa , Intolerância à Glucose/metabolismo , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Obesidade/metabolismo
15.
PLoS Genet ; 15(7): e1008292, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31339933

RESUMO

Red light promotes germination after activating phytochrome phyB, which destabilizes the germination repressor PIF1. Early upon seed imbibition, canopy light, unfavorable for photosynthesis, represses germination by stabilizing PIF1 after inactivating phyB. Paradoxically, later upon imbibition, canopy light stimulates germination after activating phytochrome phyA. phyA-mediated germination is poorly understood and, intriguingly, is inefficient, compared to phyB-mediated germination, raising the question of its physiological significance. A genetic screen identified polyamine uptake transporter 2 (put2) mutants that overaccumulate polyamines, a class of antioxidant polycations implicated in numerous cellular functions, which we found promote phyA-mediated germination. In WT seeds, our data suggest that canopy light represses polyamines accumulation through PIF1 while red light promotes polyamines accumulation. We show that canopy light also downregulates PIF1 levels, through phyA; however, PIF1 reaccumulates rapidly, which limits phyA-mediated germination. High polyamines levels in decaying seeds bypass PIF1 repression of germination and stimulate phyA-mediated germination, suggesting an adaptive mechanism promoting survival when viability is compromised.


Assuntos
1-Pirrolina-5-Carboxilato Desidrogenase/genética , Sistemas de Transporte de Aminoácidos/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fitocromo A/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , 1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação para Baixo , Germinação , Luz , Mutação , Poliaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1544-1555, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326539

RESUMO

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Biotinilação , Bortezomib/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia
17.
Biochim Biophys Acta Biomembr ; 1861(9): 1558-1567, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295473

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/fisiologia , Aminoácidos/metabolismo , Arginina/metabolismo , Sítios de Ligação , Transporte Biológico , Colesterol/metabolismo , Glutamina/metabolismo , Humanos , Transporte de Íons , Cinética , Lisossomos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
18.
Fish Physiol Biochem ; 45(5): 1589-1602, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31256306

RESUMO

The present study evaluated the influence of dietary soybean glycinin on growth performance, intestinal morphology, free intestinal amino acid (AA) content, and intestinal AA transporter (AAT) mRNA levels in juvenile grass carp (Ctenopharyngodon idella). Results were displayed as follows: (1) 8% dietary glycinin decreased growth performance, inhibited intestinal growth, and caused intestinal histology damage of grass carp; (2) dietary glycinin decreased the content of free neutral AAs including Val, Ser, Tyr, Ala, Pro, and Gln in all intestinal segments, and Thr, Ile, Leu, Phe, and Gly in the MI and DI while downregulated the mRNA levels of corresponding transporters including SLC38A2, SLC6A19b, and SLC6A14 in all intestinal segments, and SLC7A5, SLC7A8, and SLC1A5 in the MI and DI. Dietary glycinin decreased the content of free basic AAs including Arg in the MI and DI and His in all intestinal segments while downregulated cationic AAT SLC7A1 mRNA levels in the MI and DI. Dietary glycinin decreased the content of free acidic AAs including Glu in all intestinal segments and Asp in the MI and DI while decreased mRNA levels of corresponding transporters including SLC1A2a in all intestinal segments and SLC1A3 in the MI and DI; (3) the digestion trial showed that basic subunits of glycinin was hard to digest in the intestine of grass carp; (4) co-administration of glutamine with glycinin partially alleviated the negative effects. Overall, glycinin decreased intestinal AA absorption capacity partly contributed by decreased AATs' mRNA levels and the indigestibility of glycinin.


Assuntos
Aminoácidos/metabolismo , Carpas/metabolismo , Globulinas/toxicidade , Intestinos/efeitos dos fármacos , Proteínas de Soja/toxicidade , Soja/química , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Ração Animal/análise , Animais , Dieta , Digestão/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Globulinas/química , Transportador 1 de Peptídeos/genética , Transportador 1 de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Soja/química
19.
PLoS One ; 14(6): e0218806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233570

RESUMO

In the course of recent comparative genomic studies conducted on nervous systems across the phylogeny, current thinking is leaning in favor of more heterogeneity among nervous systems than what was initially expected. The isolation and characterization of molecular components that constitute the cnidarian neuron is not only of interest to the physiologist but also, on a larger scale, to those who study the evolution of nervous systems. Understanding the function of those ancient neurons involves the identification of neurotransmitters and their precursors, the description of nutrients used by neurons for metabolic purposes and the identification of integral membrane proteins that bind to those compounds. Using a molecular cloning strategy targeting membrane proteins that are known to be present in all forms of life, we isolated a member of the solute carrier family 6 from the scyphozoan jellyfish Cyanea capillata. The phylogenetic analysis suggested that the new transporter sequence belongs to an ancestral group of the nutrient amino acid transporter subfamily and is part of a cluster of cnidarian sequences which may translocate the same substrate. We found that the jellyfish transporter is expressed in neurons of the motor nerve net of the animal. To this end, we established an in situ hybridization protocol for the tissues of C. capillata and developed a specific antibody to the jellyfish transporter. Finally, we showed that the gene that codes for the jellyfish transporter also expresses a long non-coding RNA. We hope that this research will contribute to studies that seek to understand what constitutes a neuron in species that belong to an ancient phylum.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Cifozoários/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Animais , Clonagem Molecular , Evolução Molecular , Feminino , Células HEK293 , Humanos , Hibridização In Situ , Neurônios Motores/metabolismo , Rede Nervosa/metabolismo , Oócitos/metabolismo , Filogenia , RNA Longo não Codificante/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cifozoários/classificação , Cifozoários/genética , Homologia de Sequência de Aminoácidos , Xenopus
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