RESUMO
Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Coenzima A/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Desacoplamento Mitocondrial/genética , Proteínas de Desacoplamento Mitocondrial/metabolismo , Modelos Biológicos , NAD/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Plantas/genéticaRESUMO
Imbalance in nutrients can affect digestibility of amino acids by altering gene expression of amino acid transporters. We investigated digestibility and molecular transporters of essential amino acids in chickens fed a methionine-deficient diet. A total of 40 chicks (23 D old) were randomly assigned to either a control (0.49% methionine) or a deficient (0.28%) diet until 41 D when they were sampled for Pectoralis (P.) major, kidney, ileum, and hypothalamus for mRNA expression analysis. The ileal content was collected for apparent ileal digestibility (AID) analysis. Birds fed the deficient diet had reduced growth and worse feed efficiency compared to control. The AID of methionine was similar between both groups. The AID of other essential amino acids was higher in the deficient group than control. mRNA expression of b0,+ AT and LAT4 were upregulated in the ileum and kidney but LAT1 was downregulated only in kidney of the deficient group compared to control. In the P. major, SNAT1, SNAT2, and CAT1 were upregulated in the deficient group compared to control. A diet deficiency in methionine affects digestibility of essential amino acids and cysteine, but not the digestibility of methionine. The change in digestibility is reflected in the mRNA expression of amino acid transporters across different tissues.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Proteínas Aviárias/genética , Galinhas/fisiologia , Digestão/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Metionina/deficiência , Sistemas de Transporte de Aminoácidos/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Proteínas Aviárias/metabolismo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , MasculinoRESUMO
Gene regulation in yeast occurs at the transcription level, i.e. the basal level of expression is very low and increased transcription requires gene-specific transcription factors allowing the recruitment of basal transcriptional machinery. Saccharomyces cerevisiae BAP2 gene encodes the permease responsible for most uptake of leucine, valine and isoleucine, amino acids that this yeast can use as nitrogen sources. Moreover, BAP2 expression is known to be induced by the presence of amino acids such as leucine. In this context, the results presented in this paper show that BAP2 is an inducible gene in the presence of nitrogen-non-preferred source proline but exhibits high constitutive non-inducible expression in nitrogen-preferred source ammonium. BAP2 expression is regulated by the SPS sensor system and transcription factors Leu3, Gcn4 and Dal81. This can be achieved or not through a direct binding to the promoter depending on the quality of the nitrogen source. We further demonstrate here that an interaction occurs in vivo between Uga3 â the transcriptional activator responsible for γ-aminobutyric acid (GABA)-dependent induction of the GABA genes â and the regulatory region of the BAP2 gene, which leads to an increase in BAP2 transcription.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/metabolismo , Transdução de Sinais , Virulência/genética , Proteínas ras/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/metabolismo , Cryptococcus neoformans/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mutagênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Estresse Fisiológico , Temperatura , Proteínas ras/genéticaRESUMO
BACKGROUND: Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. METHODOLOGY/PRINCIPAL FINDINGS: RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. CONCLUSIONS/SIGNIFICANCE: In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.
Assuntos
Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/metabolismo , Arginase/metabolismo , Arginina/metabolismo , Leishmania/enzimologia , Macrófagos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Animais , Arginase/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , TranscriptomaRESUMO
Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Sistemas de Transporte de Aminoácidos/genética , Animais , Antifúngicos/farmacologia , Carbono/metabolismo , Criptococose/mortalidade , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glucose/metabolismo , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Nitrogênio/metabolismo , Estresse Oxidativo , Fenótipo , Especificidade por Substrato , Temperatura , Virulência/genéticaRESUMO
Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.
Assuntos
Ácidos/toxicidade , Lactobacillus delbrueckii/efeitos dos fármacos , Lactobacillus delbrueckii/enzimologia , Ornitina Descarboxilase/metabolismo , Estresse Fisiológico , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Lactobacillus delbrueckii/fisiologia , Ornitina Descarboxilase/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A descrição do transporte placentário envolve informações sobre mecanismos, propriedades e regulação genética das substâncias moleculares. As trocas materno-fetais se concretizam pelas células do sinciotrofoblasto (microvilosidades e camada basal da membrana plasmática) bem como o tecido conectivo e o endotélio capilar fetal. A transferência de glicose ocorre pelo mecanismo de difusão facilitada e já foram identificados na placenta humana aproximadamente 15 sistemas de transporte dos aminoácidos. A grande maioria das drogas é transportada pelo mecanismo de difusão, porém uma pequena parte depende de suas características fisicoquímicas.
Assuntos
Feminino , Gravidez , Transporte Biológico , Glucose/metabolismo , Placenta/metabolismo , Sangue Fetal , Sistemas de Transporte de Aminoácidos/metabolismo , Troca Materno-Fetal/fisiologia , Vitaminas/metabolismo , Retardo do Crescimento Fetal/etiologiaRESUMO
Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Antifúngicos/farmacologia , Brefeldina A/farmacologia , Leucina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Citrulina/metabolismo , Cinética , PrótonsRESUMO
Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.