RESUMO
Multivalency in lectins plays a pivotal role in influencing glycan cross-linking, thereby affecting lectin functionality. This multivalency can be achieved through oligomerization, the presence of tandemly repeated carbohydrate recognition domains, or a combination of both. Unlike lectins that rely on multiple factors for the oligomerization of identical monomers, tandem-repeat lectins inherently possess multivalency, independent of this complex process. The repeat domains, although not identical, display slightly distinct specificities within a predetermined geometry, enhancing specificity, affinity, avidity and even oligomerization. Despite the recognition of this structural characteristic in recently discovered lectins by numerous studies, a unified criterion to define tandem-repeat lectins is still necessary. We suggest defining them multivalent lectins with intrachain tandem repeats corresponding to carbohydrate recognition domains, independent of oligomerization. This systematic review examines the folding and phyletic diversity of tandem-repeat lectins and refers to relevant literature. Our study categorizes all lectins with tandemly repeated carbohydrate recognition domains into nine distinct folding classes associated with specific biological functions. Our findings provide a comprehensive description and analysis of tandem-repeat lectins in terms of their functions and structural features. Our exploration of phyletic and functional diversity has revealed previously undocumented tandem-repeat lectins. We propose research directions aimed at enhancing our understanding of the origins of tandem-repeat lectin and fostering the development of medical and biotechnological applications, notably in the design of artificial sugars and neolectins.
Assuntos
Lectinas , Sequências de Repetição em Tandem , Animais , Humanos , Lectinas/química , Lectinas/metabolismoRESUMO
Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.
Assuntos
Galectinas , Gastrópodes , Animais , Galectinas/química , Filogenia , Sulfatos , Galactosídeos/química , Gastrópodes/genética , Gastrópodes/metabolismo , Polissacarídeos , Moluscos/genética , HeparinaRESUMO
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current (If) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. If sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/metabolismo , Consumo de Oxigênio , Animais , Linhagem Celular , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/análise , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Ratos WistarRESUMO
The physiological regulation of hepatic glutathione efflux by catecholamines is poorly understood. The purpose of this work was to review the role of adrenergic receptors (AR) on total glutathione (GT) efflux in rat liver. Two models were used: isolated hepatocytes and perfused livers. In hepatocytes 10⯵M adrenaline (Adr), but not isoproterenol (Iso) a ß-AR agonist, or phenylephrine (Phe) an α1-AR agonist, (in a Krebs-Henseleit buffer (KHB) enriched with Ca2+ and some aminoacids) increased in 13% GT efflux. In livers perfused with KHB, Adr or Iso at 1 µmolar doses (but not Phe) stimulated 11-fold initial velocity of GT release, but only during the first 2â¯min of perfusion. This immediate response progressively disappeared during the following 15â¯min of perfusion. A second phase of GT efflux, observed between 2 and 14â¯min of perfusion, mimics the one reported earlier in isolated hepatocytes. The ED50 for Adr and Iso activation are in the range of 320â¯nM and 10â¯nM, respectively. Iso-mediated GT release requires Ca2+ to work, and was prevented by H89, glibenclamide, cystic fibrosis transmembrane regulator (CFTR) antibodies, and a direct CFTR inhibitor. This short-lived GT release system is associated to PKA activation and probably operates through CFTR.
Assuntos
Glutationa/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Hepatócitos/citologia , Isoproterenol/farmacologia , Fígado/citologia , Masculino , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
Triosephosphate isomerase (TIM) is a ubiquitous enzyme, which appeared early in evolution. TIM is responsible for obtaining net ATP from glycolysis and producing an extra pyruvate molecule for each glucose molecule, under aerobic and anaerobic conditions. It is placed in a metabolic crossroad that allows a quick balance of the triose phosphate aldolase produced by glycolysis, and is also linked to lipid metabolism through the alternation of glycerol-3-phosphate and the pentose cycle. TIM is one of the most studied enzymes with more than 199 structures deposited in the PDB. The interest for this enzyme stems from the fact that it is involved in glycolysis, but also in aging, human diseases and metabolism. TIM has been a target in the search for chemical compounds against infectious diseases and is a model to study catalytic features. Until February 2017, 62% of all residues of the protein have been studied by mutagenesis and/or using other approaches. Here, we present a detailed and comprehensive recompilation of the reported effects on TIM catalysis, stability, druggability and human disease produced by each of the amino acids studied, contributing to a better understanding of the properties of this fundamental protein. The information reviewed here shows that the role of the noncatalytic residues depend on their molecular context, the delicate balance between the short and long-range interactions in concerted action determining the properties of the protein. Each protein should be regarded as a unique entity that has evolved to be functional in the organism to which it belongs. Proteins 2017; 85:1190-1211. © 2017 Wiley Periodicals, Inc.
Assuntos
Inibidores Enzimáticos/química , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Estabilidade Enzimática , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
BACKGROUND: Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s. RESULTS: We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis. CONCLUSION: Our findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Betaína/análogos & derivados , Evolução Molecular , Osmose , Spinacia oleracea/enzimologia , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/química , Biocatálise , Estabilidade Enzimática , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Oxirredução , FilogeniaRESUMO
Renal iron overload is a common complication of diabetes that leads to oxidative stress and mitochondrial dysfunction in the kidneys. This study investigated the effects of iron chelation using deferiprone on mitochondrial dysfunction and oxidative stress in the renal cortex of a murine model of type 2 diabetes. Diabetic rats were treated with deferiprone (50 mg/kg BW) for 16 weeks. Our results show that iron chelation with deferiprone significantly increased the nuclear accumulation of Nrf2, a transcription factor that regulates the expression of antioxidant enzymes. This led to enhanced antioxidant capacity, reduced production of reactive oxygen species, and improved mitochondrial bioenergetic function in diabetic rats. However, chronic iron chelation led to altered mitochondrial respiration and increased oxidative stress in non-diabetic rats. In conclusion, our findings suggest that iron chelation with deferiprone protects mitochondrial bioenergetics and mitigates oxidative stress in the renal cortex, involving the NRF2 pathway in type 2 diabetes.
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BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) -aspirin, naproxen, nimesulide, and piroxicam- lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. RESULTS: Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. CONCLUSIONS: NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.
Assuntos
Adipócitos/enzimologia , Anti-Inflamatórios não Esteroides/farmacologia , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Peróxido de Hidrogênio/metabolismo , Lipólise/efeitos dos fármacos , NADPH Oxidases/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Aquaporinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Masculino , NADPH Oxidases/antagonistas & inibidores , Ratos , Ratos Wistar , Nitrato de Prata/farmacologiaRESUMO
The Bacillus subtilis strain 168 genome contains the chr3N-chr3C genes encoding the Chr3N/Chr3C protein pair of the chromate ion transporter (CHR) superfamily. Chr3N/Chr3C confers chromate resistance in Escherichia coli only when both proteins are expressed. Upstream of chr3N is the chrS gene encoding ChrS, a protein with homology to the Lrp/AsnC family of transcriptional regulators. When the chrS-chr3N-chr3C gene cluster was transferred to E. coli, a diminished level of chromate resistance was observed, as compared with E. coli transformants bearing only the chromate resistance genes, which displayed full resistance. These data suggested that the chrS gene product acts as negative regulator. RT-PCR assays demonstrated that expression of chrS diminishes transcription of the chromate resistance genes in E. coli, and that this repression was overcome by chromate. Electrophoretic mobility shift assays showed that purified ChrS protein specifically binds to the 5' region of chrS. These results indicate that the chr gene cluster forms an operon regulated negatively by ChrS binding to its own gene's regulatory region, and positively by chromate ions. Sequence analysis revealed similar operons in many Bacillales strains, suggesting some adaptive advantage. This is the first example of a bacterial heavy-metal resistance system controlled by an Lrp-type transcriptional regulator.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Cromatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Cromatos/toxicidade , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Proteína Reguladora de Resposta a Leucina/genética , Família Multigênica , Óperon , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In most eukaryotes the subunit 2 of cytochrome c oxidase (COX2) is encoded in intact mitochondrial genes. Some green algae, however, exhibit split cox2 genes (cox2a and cox2b) encoding two polypeptides (COX2A and COX2B) that form a heterodimeric COX2 subunit. Here, we analyzed the distribution of intact and split cox2 gene sequences in 39 phylogenetically diverse green algae in phylum Chlorophyta obtained from databases (28 sequences from 22 taxa) and from new cox2 data generated in this work (23 sequences from 18 taxa). Our results support previous observations based on a smaller number of taxa, indicating that algae in classes Prasinophyceae, Ulvophyceae, and Trebouxiophyceae contain orthodox, intact mitochondrial cox2 genes. In contrast, all of the algae in Chlorophyceae that we examined exhibited split cox2 genes, and could be separated into two groups: one that has a mitochondrion-localized cox2a gene and a nucleus-localized cox2b gene ("Scenedesmus-like"), and another that has both cox2a and cox2b genes in the nucleus ("Chlamydomonas-like"). The location of the split cox2a and cox2b genes was inferred using five different criteria: differences in amino acid sequences, codon usage (mitochondrial vs. nuclear), codon preference (third position frequencies), presence of nucleotide sequences encoding mitochondrial targeting sequences and presence of spliceosomal introns. Distinct green algae could be grouped according to the form of cox2 gene they contain: intact or fragmented, mitochondrion- or nucleus-localized, and intron-containing or intron-less. We present a model describing the events that led to mitochondrial cox2 gene fragmentation and the independent and sequential migration of cox2a and cox2b genes to the nucleus in chlorophycean green algae. We also suggest that the distribution of the different forms of the cox2 gene provides important insights into the phylogenetic relationships among major groups of Chlorophyceae.
Assuntos
Núcleo Celular/genética , Clorófitas/genética , Ciclo-Oxigenase 2/genética , Modelos Genéticos , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Códon/genética , Biologia Computacional , Primers do DNA/genética , DNA Mitocondrial/genética , Funções Verossimilhança , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
In addition to playing a central role in the mitochondria as the main producer of ATP, FOF1-ATP synthase performs diverse key regulatory functions in the cell membrane. Its malfunction has been linked to a growing number of human diseases, including hypertension, atherosclerosis, cancer, and some neurodegenerative, autoimmune, and aging diseases. Furthermore, inhibition of this enzyme jeopardizes the survival of several bacterial pathogens of public health concern. Therefore, FOF1-ATP synthase has emerged as a novel drug target both to treat human diseases and to combat antibiotic resistance. In this work, we carried out a computational characterization of the binding sites of the fungal antibiotic aurovertin in the bovine F1 subcomplex, which shares a large identity with the human enzyme. Molecular dynamics simulations showed that although the binding sites can be described as preformed, the inhibitor hinders inter-subunit communications and exerts long-range effects on the dynamics of the catalytic site residues. End-point binding free energy calculations revealed hot spot residues for aurovertin recognition. These residues were also relevant to stabilize solvent sites determined from mixed-solvent molecular dynamics, which mimic the interaction between aurovertin and the enzyme, and could be used as pharmacophore constraints in virtual screening campaigns. To explore the possibility of finding species-specific inhibitors targeting the aurovertin binding site, we performed free energy calculations for two bacterial enzymes with experimentally solved 3D structures. Finally, an analysis of bacterial sequences was carried out to determine conservation of the aurovertin binding site. Taken together, our results constitute a first step in paving the way for structure-based development of new allosteric drugs targeting FOF1-ATP synthase sites of exogenous inhibitors.
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Oxidative stress is a factor that contributes to the development of complications in diabetes; however, its effects can be counteracted using exogenous antioxidants that are found in some plants, which is why people turn to traditional medicines in the search for therapeutic treatment. Justicia spicigera has been demonstrated to have the capacity to reduce glycemic levels; however, its effects on non-insulin-dependent organs such as the liver have not been reported. During 30 days of administration of Justicia spicigera ethanol extract, the blood glucose and weight of rats were measured every 5 days. Once the treatment was concluded, the rats were sacrificed. Corporal weight, blood glucose, cholesterol, very-low-density lipoprotein (VLDL), triglycerides, total lipids, and liver profile were reduced in the diabetic condition and normalized with the application of ethanol extract from J. spicigera (EJS). Additionally, there was a significant increase in catalase and superoxide dismutase activity in the control diabetic rats, a decrease in their activity with the extract administration, and no effect on normoglycemic rats. In conclusion, EJS is considered to be capable of reducing oxidative stress by maintaining diminished lipid and liver function profiles in male Wistar rats with streptozotocin-induced diabetes.
Assuntos
Diabetes Mellitus Experimental , Justicia , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Glicemia , Diabetes Mellitus Experimental/tratamento farmacológico , Etanol/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Masculino , Estresse Oxidativo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos , Ratos Wistar , EstreptozocinaRESUMO
Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.
Assuntos
Molibdênio/metabolismo , Sulfatos/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Ânions/metabolismo , Bactérias/metabolismo , Transporte Biológico , Cromatos/metabolismo , Ácido Selênico , Compostos de Selênio/metabolismo , Compostos de Tungstênio/metabolismoRESUMO
Cardiovascular diseases (CVD) (such as occlusion of the coronary arteries, hypertensive heart diseases and strokes) are diseases that generate thousands of patients with a high mortality rate worldwide. Many of these cardiovascular pathologies, during their development, generate a state of oxidative stress that leads to a deterioration in the patient's conditions associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Within these reactive species we find superoxide anion (O2â¢-), hydroxyl radical (â¢OH), nitric oxide (NOâ¢), as well as other species of non-free radicals such as hydrogen peroxide (H2O2), hypochlorous acid (HClO) and peroxynitrite (ONOO-). A molecule that actively participates in counteracting the oxidizing effect of reactive species is reduced glutathione (GSH), a tripeptide that is present in all tissues and that its synthesis and/or regeneration is very important to be able to respond to the increase in oxidizing agents. In this review, we will address the role of glutathione, its synthesis in both the heart and the liver, and its importance in preventing or reducing deleterious ROS effects in cardiovascular diseases.
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G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.
Assuntos
Motivos de Aminoácidos , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Oócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Sequência Conservada , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Oócitos/efeitos dos fármacos , Filogenia , Ligação Proteica , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Reactive oxygen species participate in regulating intracellular signaling pathways. Herein, we investigated the reported opposite effects of hydrogen peroxide (H2 O2 ) on metabolic signaling mediated by activated α1 - and ß-adrenoceptors (ARs) in hepatocytes. In isolated rat hepatocytes, stimulation of α1 -AR increases H2 O2 production via NADPH oxidase 2 (NOX2) activation. We find that the H2 O2 thus produced is essential for α1 -AR-mediated activation of the classical hepatic glycogenolytic, gluconeogenic, and ureagenic responses. However, H2 O2 inhibits ß-AR-mediated activation of these metabolic responses. We show that H2 O2 mediates its effects on α1 -AR and ß-AR by permeating cells through aquaporin 8 (AQP8) channels and promoting Ca2+ mobilization. Thus, our findings reveal a novel NOX2-H2 O2 -AQP8-Ca2+ signaling cascade acting downstream of α1 -AR in hepatocytes, which, by negatively regulating ß-AR signaling, establishes negative crosstalk between the two pathways.
Assuntos
Aquaporinas/metabolismo , Hepatócitos/metabolismo , Peróxido de Hidrogênio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Animais , Sinalização do Cálcio , Gluconeogênese , Glicogenólise , Humanos , Masculino , NADPH Oxidase 2/metabolismo , Ratos , Ratos WistarRESUMO
Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.
Assuntos
Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Cromatos/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatos/farmacologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Ordem dos Genes , Íons/metabolismo , Íons/farmacologia , Proteínas de Membrana Transportadoras/genética , Óperon , Transcrição GênicaRESUMO
Aldehyde dehydrogenases (ALDHs) comprise one of the most ancient protein superfamilies widely distributed in the three domains of life. Their members have been extensively studied in animals and plants, sorted out in different ALDH protein families and their participation in a broad variety of metabolic pathways has been documented. Paradoxically, no systematic studies comprising ALDHs from bacteria have been performed so far. Among bacteria, the genus Pseudomonas occupies numerous ecological niches, and is one of the most complex bacterial genera with the largest number of known species. For these reasons, we selected Pseudomonas as a paradigm to analyze the diversity of ALDHs in bacteria. With this aim, complete Pseudomonas genome sequences and annotations were retrieved from NCBI's RefSeq genome database. The 258 Pseudomonas strains belong to 46 different species, along with 23 with no species designation. The genomes of these Pseudomonas contain from 3,315 to 6,825 annotated protein coding genes. A total of 6,510 ALDH sequences were found in the selected Pseudomonas, with a median of 24 ALDH-coding genes per strain (by comparison humans possess only 19 different ALDH loci). Pseudomonas saudiphocaensis possesses the lowest number of aldh genes (9), while Pseudomonas pseudoalcaligenes KF707 NBRC110670 possesses the maximum number of aldh genes (49). The ALDHs found in Pseudomonas can be sorted out into 42 protein families, with a predominance of 14 families, which contained 76% of all ALDHs found. In this regard, it is important to note that many Pseudomonas genomes have multiple aldh genes coding for proteins belonging to the same family. Given that all strains contained members of families ALDH4, ALDH5, ALDH6, ALDH14, ALDH18 and ALDH27, we consider these families to be part of the core Pseudomonas genome.
Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Pseudomonas/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Análise por Conglomerados , Humanos , Metabolômica , Proteômica , Pseudomonas/genética , Retinal Desidrogenase , Células Tumorais CultivadasRESUMO
Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.
Assuntos
Amaranthaceae/genética , Oxigenases/genética , Filogenia , Proteínas de Plantas/genética , Amaranthaceae/química , Sequência de Aminoácidos , Sequência Conservada , Evolução Molecular , Simulação de Acoplamento Molecular , Oxigenases/química , Proteínas de Plantas/química , Domínios Proteicos , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Membrane fatty acid composition has an important role in yeast stress resistance, particularly in temperature tolerance. Most studies investigating temperature and membrane fatty acids use the yeast Saccharomyces cerevisiae without considering other yeasts, such as Kluyveromyces marxianus, which has physiological differences and industrial advantages with respect to S. cerevisiae. One of the primary traits of K. marxianus is its thermotolerance. The effect of fatty acid addition (oleic acid, linoleic acid, linolenic acid and araquidic acid) on the thermotolerance of the K. marxianus strain SLP1 was evaluated. SLP1 yeast exhibited temperature tolerance of up to 50°C; at 55°C, viability was reduced significantly, probably due to an increase in the generation of reactive oxygen chemical species. Externally added fatty acids were incorporated in the yeast membrane, increasing their proportion to approximately 70%, thereby changing membrane fluidity. SLP1 cells supplemented with polyunsaturated fatty acids decreased cell thermotolerance and increased the degree of lipoperoxidation, while arachidic acid addition exhibited a tendency to increase yeast thermotolerance.