RESUMEN
Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a ß-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble ß-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity. KEY POINTS: ⢠Aromatic residues in the BD are involved in BD and CD folding. ⢠Aromatic residues in the BD near the CD active site modulate enzyme activity. ⢠BD interacts with CD and closely modulates enzyme activity.
Asunto(s)
Dominio Catalítico , Pliegue de Proteína , Trehalasa , Trehalasa/genética , Trehalasa/metabolismo , Trehalasa/química , Aminoácidos Aromáticos/metabolismo , Sustitución de AminoácidosRESUMEN
In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.
Asunto(s)
Fimbrias Bacterianas , Osmorregulación , Trehalosa , Vejiga Urinaria , Infecciones Urinarias , Animales , Trehalosa/metabolismo , Ratones , Vejiga Urinaria/microbiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Infecciones Urinarias/microbiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Animales de Enfermedad , Femenino , Presión Osmótica , Escherichia coli Patógena Extraintestinal/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Urea/metabolismo , Trehalasa/metabolismo , Trehalasa/genética , Eliminación de Gen , Glucosa/metabolismoRESUMEN
The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs.
Asunto(s)
Bombyx , Ecdisteroides , Hormonas de Insectos , Proteínas de Insectos , Larva , Animales , Bombyx/genética , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Bombyx/enzimología , Ecdisteroides/metabolismo , Hormonas de Insectos/metabolismo , Hormonas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Larva/genética , Trehalasa/metabolismo , Trehalasa/genética , Transducción de Señal , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Monosacáridos/genéticaRESUMEN
Trehalose serves as a primary circulatory sugar in insects which is crucial in energy metabolism and stress recovery. It is hydrolyzed into two glucose molecules by trehalase. Silencing or inhibiting trehalase results in reduced fitness, developmental defects, and insect mortality. Despite its importance, the molecular response of insects to trehalase inhibition is not known. Here, we performed transcriptomic analyses of Helicoverpa armigera treated with validamycin A (VA), a trehalase inhibitor. VA ingestion resulted in increased mortality, developmental delay, and reduced ex vivo trehalase activity. Pathway enrichment and gene ontology analyses suggest that key genes involved in carbohydrate, protein, fatty acid, and mitochondria-related metabolisms are deregulated. The activation of protein and fat degradation may be necessary to fulfil energy requirements, evidenced by the dysregulated expression of critical genes in these metabolisms. Co-expression analysis supports the notion that trehalase inhibition leads to putative interaction with key regulators of other pathways. Metabolomics correlates with transcriptomics to show reduced levels of key energy metabolites. VA generates an energy-deficient condition, and insects activate alternate pathways to facilitate the energy demand. Overall, this study provides insights into the molecular mechanisms underlying the response of insects to trehalase inhibition and highlights potential targets for insect control.
Asunto(s)
Metabolismo Energético , Trehalasa , Animales , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Perfilación de la Expresión Génica , Helicoverpa armigera , Inositol/farmacología , Inositol/metabolismo , Inositol/análogos & derivados , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva , Transcriptoma/genética , Trehalasa/metabolismo , Trehalasa/genética , Trehalasa/antagonistas & inhibidores , Trehalosa/metabolismoRESUMEN
Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.
Asunto(s)
Fumonisinas , Proteínas Fúngicas , Fusarium , Trehalasa , Fusarium/metabolismo , Fusarium/efectos de los fármacos , Fusarium/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Fumonisinas/metabolismo , Trehalasa/genética , Trehalasa/metabolismo , Trehalasa/química , Trehalasa/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Inositol/análogos & derivados , Inositol/farmacología , Inositol/química , Enfermedades de las Plantas/microbiología , Antifúngicos/farmacología , Antifúngicos/química , Streptomyces/metabolismo , Streptomyces/genética , Streptomyces/químicaRESUMEN
Trehalase gene is mainly expressed in the digestive circulatory system for regulating energy metabolism and chitin synthesis in insects, but it is significantly expressed in gill for immunomodulation in shrimp. However, its function in regulating immunity, particularly metal resistance in crustaceans has yet to be elucidated. In this study, one Tre2 gene (NdTre2) was isolated from Neocaridina denticulata sinensis. It could bind to Cd2+ and inhibit its toxicity. Spatiotemporal expression analysis showed that the expression of NdTre2 was highest in the gill and significantly reduced at 12 h after Cd2+ stimulation. The transcriptomic analysis of the gill after NdTre2 knockdown showed that the expression of genes synthetizing 20E was up-regulated and the increased 20E could further induce apoptosis by activating the intrinsic mitochondrial pathway, exogenous death receptor-ligand pathway, and MAPK pathway. In vitro, overexpressing NdTre2 enhanced the tolerance of E. coli in Cd2+ environment. In summary, these results indicate that NdTre2 plays an essential role in regulating immunity and chitin metabolism in N. denticulata sinensis.
Asunto(s)
Apoptosis , Cadmio , Trehalasa , Cadmio/toxicidad , Animales , Apoptosis/efectos de los fármacos , Trehalasa/metabolismo , Trehalasa/genética , Contaminantes Químicos del Agua/toxicidad , Decápodos/fisiología , Decápodos/genéticaRESUMEN
The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) µmolmg-1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) µmolmg-1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.
Asunto(s)
Frío , Comamonadaceae , Trehalasa , Trehalasa/metabolismo , Trehalasa/genética , Trehalasa/química , Especificidad por Sustrato , Comamonadaceae/enzimología , Comamonadaceae/genética , Dominio Catalítico , Trehalosa/metabolismo , Trehalosa/farmacología , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Secuencia de Aminoácidos , Estabilidad de Enzimas , Adaptación FisiológicaRESUMEN
Trehalases (TREs) are pivotal enzymes involved in insect development and reproduction, making them prime targets for pest control. We investigated the inhibitory effect of three thiazolidinones with piperine skeletons (6a, 7b, and 7e) on TRE activity and assessed their impact on the growth and development of the fall armyworm (FAW), Spodoptera frugiperda. The compounds were injected into FAW larvae, while the control group was treated with 2% DMSO solvent. All three compounds effectively inhibited TRE activity, resulting in a significant extension of the pupal development stage. Moreover, the treated larvae exhibited significantly decreased survival rates and a higher incidence of abnormal phenotypes related to growth and development compared to the control group. These results suggest that these TRE inhibitors affect the molting of larvae by regulating the chitin metabolism pathway, ultimately reducing their survival rates. Consequently, these compounds hold potential as environmentally friendly insecticides.
Asunto(s)
Alcaloides , Benzodioxoles , Insecticidas , Piperidinas , Alcamidas Poliinsaturadas , Trehalasa , Animales , Larva , Spodoptera , Trehalasa/genética , Insecticidas/farmacologíaRESUMEN
Insect trehalases have been identified as promising new targets for pest control. These key enzymes are involved in trehalose hydrolysis and plays an important role in insect growth and development. In this contribution, plant and microbial compounds, namely validamycin A, amygdalin, and phloridzin, were evaluated for their effect, through trehalase inhibition, on Acyrthosiphon pisum aphid. The latter is part of the Aphididae family, main pests as phytovirus vectors and being very harmful for crops. Validamycin A was confirmed as an excellent trehalase inhibitor with an half maximal inhibitory concentration and inhibitor constant of 2.2 × 10-7 and 5 × 10-8 M, respectively, with a mortality rate of ~80% on a A. pisum population. Unlike validamycin A, the insect lethal efficacy of amygdalin and phloridzin did not correspond to their trehalase inhibition, probably due to their hydrolysis by insect ß-glucosidases. Our docking studies showed that none of the three compounds can bind to the trehalase active site, unlike their hydrolyzed counterparts, that is, validoxylamine A, phloretin, and prunasin. Validoxylamine A would be by far the best trehalase binder, followed by phloretin and prunasin.
Asunto(s)
Áfidos , Trehalasa , Animales , Amigdalina , Áfidos/efectos de los fármacos , Áfidos/enzimología , Inositol/análogos & derivados , Nitrilos , Floretina , Florizina , Trehalasa/antagonistas & inhibidoresRESUMEN
Trehalase has attracted widespread attention in medicine, agriculture, food, and ethanol industry due to its ability to specifically degrade trehalose. Efficient expression of trehalase remains a challenge. In this study, a putative trehalase-encoding gene (Tre-zm) from Zunongwangia mangrovi was explored using gene-mining strategy and heterologously expressed in E. coli. Trehalase activity reached 3374 U·mL-1 after fermentation optimization. The scale-up fermentation in a 15 L fermenter was achieved with a trehalase production of 15,068 U·mL-1. The recombinant trehalase TreZM was purified and characterized. It displayed optimal activity at 35 °C and pH 8.5, with Mn2+, Sn2+, Na+, and Fe2+ promoting the activity. Notably, TreZM showed significant inhibition effect on biofilm forming of Staphylococcus epidermidis. The combination of TreZM with a low concentration of antibiotics could inhibit 70 % biofilm formation of Staphylococcus epidermidis and 28 % of Pseudomonas aeruginosa. Hence, this study provides a promising candidate for industrial production of trehalase and highlights its potential application to control harmful biofilms.
Asunto(s)
Escherichia coli , Trehalasa , Trehalasa/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Trehalosa/farmacología , Trehalosa/metabolismo , BiopelículasRESUMEN
BACKGROUND: Insects utilize trehalases (TREs) to regulate energy metabolism and chitin biosynthesis, which are essential for their growth, development, and reproduction. TREs can therefore be used as potential targets for future insecticide development. However, the roles of TREs in Frankliniella occidentalis (Pergande), a serious widespread agricultural pest, remain unclear. RESULTS: Three TRE genes were identified in F. occidentalis and cloned, and their functions were then investigated via feeding RNA interference (RNAi) and virus-induced gene silencing (VIGS) assays. The results showed that silencing FoTRE1-1 or FoTRE1-2 significantly decreased expression levels of FoGFAT, FoPGM, FoUAP, and FoCHS, which are members of the chitin biosynthesis pathway. Silencing FoTRE1-1 or FoTRE2 significantly down-regulated FoPFK and FoPK, which are members of the energy metabolism pathway. These changes resulted in 2-fold decreases in glucose and glycogen content, 2-fold increases in trehalose content, and 1.5- to 2.0-fold decreases in chitinase activity. Furthermore, knocking down FoTRE1-1 or FoTRE1-2 resulted in deformed nymphs and pupae as a result of hindered molting. The VIGS assay for the three FoTREs revealed that FoTRE1-1 or FoTRE2 caused shortened ovarioles, and reduced egg-laying and hatching rates. CONCLUSION: The results suggest that FoTRE1-1 and FoTRE1-2 play important roles in the growth and development of F. occidentalis, while FoTRE1-1 and FoTRE2 are essential for its reproduction. These three genes could be candidate targets for RNAi-based management and control of this destructive agricultural pest. © 2024 Society of Chemical Industry.
Asunto(s)
Proteínas de Insectos , Interferencia de ARN , Trehalasa , Animales , Trehalasa/genética , Trehalasa/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Ninfa/genética , Ninfa/crecimiento & desarrollo , Ninfa/enzimología , Ninfa/metabolismoRESUMEN
Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.
Asunto(s)
Bombyx , Microsporidiosis , Nosema , Animales , Trehalasa/genética , Trehalasa/metabolismo , Esporas Fúngicas/metabolismo , Nosema/genética , Bombyx/parasitologíaRESUMEN
Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.
Asunto(s)
Acremonium , Ascomicetos , Trehalasa , Verticillium , Trehalasa/genética , Virulencia/genética , Gossypium/genéticaRESUMEN
BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.
Asunto(s)
Animales , Ratones , Trehalasa/metabolismo , Virulencia/genética , Candida glabrata/genética , Trehalasa/fisiología , Trehalasa/genética , Trehalosa/análisis , Virulencia/fisiología , Candidiasis , Eliminación de Gen , Candida glabrata/fisiología , Candida glabrata/metabolismo , Candida glabrata/patogenicidad , Genes Fúngicos , HidrolasasRESUMEN
Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 æmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70 percent of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast
Asunto(s)
Candida , Maltosa , Trehalasa , Trehalosa , Candida , División Celular , Medios de Cultivo , Factores de TiempoRESUMEN
Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Proteínas Quinasas Dependientes de AMP Cíclico , Saccharomyces cerevisiae , Trehalasa , Activación Enzimática , Saccharomyces cerevisiaeRESUMEN
1. Trehalase was partially purified from Escherichia coli and characterized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C. 2. Trehalase represented approximately 50 per cent of the total protein released by osmotic shock. The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others. 3. The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol. 4. Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel). Retention of total catalytic activity averaged 32 per cent . 5. The reactor, stored for one month at -5 degrees C, retained 98 per cent of its initial immobilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose
Asunto(s)
Enzimas Inmovilizadas/aislamiento & purificación , Escherichia coli/enzimología , Trehalasa/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Enzimas Inmovilizadas/metabolismo , Calor , Dióxido de Silicio , Factores de Tiempo , Trehalasa/metabolismo , Trehalosa/análisisRESUMEN
1. Activation of Saccharomyces cerevisiae trehalase by heat shock was shown in all strains tested, including mutants in which the reponse to a glucose signal was absent. A low concentration of cAMP favored the response as seen in 2nd log cells or in ras2 and cyr1ts mutant strains. The heat shock effect upon trehalse activity was not observed under conditions of catabolite repession. 2 Neither hexokinase PII nor the heat shock protein hsp26 seemed to be involve in the axtivation of trehalase by heat shock. However, mutant strains deleted in the polyubiquitin gene showed only a 2-fold activation of the enzyme while in control strains a 5-to 7-fold irreversible activation was observed. 3. An alternative mechanism of trehalase activation by removal of an inhibitor through ligation with ubiquitin is discussed. Activation by cAMP-independent phosphorylation is also considered
Asunto(s)
Proteínas de Choque Térmico/fisiología , Saccharomyces cerevisiae/enzimología , Trehalasa/metabolismo , Activación Enzimática , Medios de Cultivo , AMP Cíclico/metabolismo , Glucosa/metabolismo , Hexoquinasa/metabolismo , Transducción de Señal , Ubiquitina/fisiologíaRESUMEN
Se realizó un experimento para evaluar el efecto de la ingestión de pan integral sobre la actividad disacaridásica intestinal. Se utilizó un total de 21 ratas macho, las cuales se agruparon según: a) dieta control con caseína más metionina, b) dieta con blanco, y c) dieta con pan integral. Despúes del periodo experimental de 10 días, se determinó la actividad específica de lactasa, mitasa, sacarasa y trealasa en distintos niveles de localización en la microvellosidad. Todas las enzimas presentaron una disminución significativa (p<0,01) de su actividad en la fracción luminal en las ratas alimentadas con pan integral. Sólo la lactasa y la maltasa mostraron una disminución de su actividad (p<0.01) en la fracción de membrana para dicha dieta. La fracción enterocitaria no mostró diferencia cuando se comparó con la dieta de pan blanco. En todos los nivles de localización la actividad disacaridásica fue mayor en la dieta control (p<0.01). Los resultados obtenidos sugieren que el efecto por "arrastre mecánico" de la fibra dietética contenida en el pan integral es el fundamental en la interacciòn fibra-actividad disacaridásica y que, por tanto, su presencia en el intestino no afecta sensiblemente la biosíntesis de dichas enzimas en el enterocito