RESUMEN
Candida glabrata and Candida albicans, the most frequently isolated candidiasis species in the world, have developed mechanisms of resistance to treatment with azoles. Among the clinically used antifungal drugs are statins and other compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), resulting in decreased growth and ergosterol levels in yeasts. Ergosterol is a key element for the formation of the yeast cell membrane. However, statins often cause DNA damage to yeast cells, facilitating mutation and drug resistance. The aim of the current contribution was to synthesize seven series of compounds as inhibitors of the HMGR enzyme of Candida ssp., and to evaluate their effect on cellular growth, ergosterol synthesis and generation of petite mutants of C. glabrata and C. albicans. Compared to the reference drugs (fluconazole and simvastatin), some HMGR inhibitors caused lower growth and ergosterol synthesis in the yeast species and generated fewer petite mutants. Moreover, heterologous expression was achieved in Pichia pastoris, and compounds 1a, 1b, 6g and 7a inhibited the activity of recombinant CgHMGR and showed better binding energy values than for α-asarone and simvastatin. Thus, we believe these are good candidates for future antifungal drug development.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Candida albicans , Candida glabrata/genética , Antifúngicos/farmacología , Simvastatina/farmacología , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes , Oxidorreductasas , Ergosterol/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Sucrose non-fermenting 1 (SNF1)-related protein kinase 1.1 (SnRK1.1; also known as KIN10 or SnRK1α) has been identified as the catalytic subunit of the complex SnRK1, the Arabidopsis thaliana homologue of a central integrator of energy and stress signalling in eukaryotes dubbed AMPK/Snf1/SnRK1. A nuclear localization of SnRK1.1 has been previously described and is in line with its function as an integrator of energy and stress signals. Here, using two biological models (Nicotiana benthamiana and Arabidopsis thaliana), native regulatory sequences, different microscopy techniques, and manipulations of cellular energy status, it was found that SnRK1.1 is localized dynamically between the nucleus and endoplasmic reticulum (ER). This distribution was confirmed at a spatial and temporal level by co-localization studies with two different fluorescent ER markers, one of them being the SnRK1.1 phosphorylation target HMGR. The ER and nuclear localization displayed a dynamic behaviour in response to perturbations of the plastidic electron transport chain. These results suggest that an ER-associated SnRK1.1 fraction might be sensing the cellular energy status, being a point of crosstalk with other ER stress regulatory pathways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/citología , Cloroplastos/metabolismo , Transporte de Electrón , Metabolismo Energético , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico , Nicotiana/citología , Nicotiana/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Biología Computacional , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/química , Unión Proteica/genética , Conformación Proteica , Secuencia de Aminoácidos/genética , Sitios de Unión , Dominio Catalítico/genética , Humanos , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/genética , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/ultraestructura , Fosforilación/genética , Especificidad por SustratoRESUMEN
Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8â¯kb hmgr1 and 0.6â¯kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs.
Asunto(s)
Hevea , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes , Látex/biosíntesis , Proteínas de Plantas , Plantas Modificadas Genéticamente , Hevea/genética , Hevea/metabolismo , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/biosíntesis , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Monoterpenes have multiple pharmacological effects on the metabolism of mevalonate. Geraniol, a dietary monoterpene, has in vitro and in vivo anti-tumor activity against several cell lines. We have studied the effects of geraniol on growth, fatty-acid metabolism, and mevalonate metabolism in the human hepatocarcinoma cell line Hep G2. Up to 100 micromol geraniol/L inhibited the growth rate and 3-hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) reductase activity of these cells. At the same concentrations, it increased the incorporation of cholesterol from the medium in a dose-dependent manner. Geraniol-treated cells incorporated less 14C-acetate into nonsaponifiable lipids, inhibiting its incorporation into cholesterol but not into squalene and lanosterol. This is indicative of an inhibition in cholesterol synthesis at a step between lanosterol and cholesterol, a fact confirmed when cells were incubated with 3H-mevalonate. The incorporation of 3H-mevalonate into protein was also inhibited, whereas its incorporation into fatty acid increased. An inhibition of delta5 desaturase activity was demonstrated by the inhibition of the conversion of 14C-dihomo-gamma-linolenic acid into arachidonic acid. Geraniol has multiple effects on mevalonate and lipid metabolism in Hep G2 cells, affecting cell proliferation. Although mevalonate depletion is not responsible for cellular growth, it affects cholesterogenesis, protein prenylation, and fatty-acid metabolism.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Ácidos Grasos/metabolismo , Ácido Mevalónico/metabolismo , Terpenos/farmacología , Acetatos/metabolismo , Monoterpenos Acíclicos , Radioisótopos de Carbono , Línea Celular , Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Grasas/metabolismo , Humanos , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/metabolismo , Ácido Mevalónico/análogos & derivados , Fosfolípidos/metabolismo , Fósforo/metabolismo , Células Tumorales CultivadasRESUMEN
Untreated maternal hypothyroidism (hypoT) has serious consequences in offspring development that may result from the effect on lactation of maternal metabolism dysfunction. We studied the effects of prolonged propylthiouracil (PTU)-induced hypoT (0.1% PTU in drinking water starting 8 days before mating until day 21 of pregnancy or for 30 days in virgin rats) on liver and mammary lipid metabolism and serum lipid concentrations. In virgins, hypoT reduced hepatic mRNAs associated with triglyceride (TG) and cholesterol synthesis (including fatty acid synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase), and induced lobuloalveolar mammary development. Pregnancy increased hepatic mRNAs associated with TG and cholesterol synthesis and uptake (including LDL receptor) and with lipid oxidation, such as acyl CoA oxidase. HypoT decreased mRNAs and the activity of proteins associated with TG synthesis, and mRNAs associated with cholesterol uptake and lipid oxidation. Pregnancy increased mammary mRNAs related to lipid oxidation and decreased cholesterol synthesis, whereas hypoT decreased mRNAs and activities of proteins associated with TG synthesis and decreased epithelial mammary tissue. Virgin and pregnant hypoT rats had increased circulating VLDL + LDL cholesterol. HypoT decreased circulating TGs in pregnant rats. The observed effects of hypoT may result in decreased mammary lipid availability. This, along with the decreased epithelial mammary tissue during lactogenesis, may contribute to the future lactational deficit of hypoT mothers.
Asunto(s)
Hipotiroidismo/patología , Hígado/metabolismo , Animales , Colesterol/metabolismo , Ácido Graso Sintasas/metabolismo , Femenino , Glucosa/metabolismo , Hormonas/metabolismo , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/metabolismo , Metabolismo de los Lípidos , Glándulas Mamarias Animales , Embarazo , Complicaciones del Embarazo , Preñez , Propiltiouracilo/farmacología , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de LDL/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Agua/químicaRESUMEN
Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activities and cholesterol content in the liver of athymic mice either bearing or not an implanted human lung mucoepidormoid carcinoma (HLMC) and in the neoplasic tissue, were analyzed. The properties of the HMG-CoA reductase of HLMC grown in nude mice and those ones found in the liver of these animals, sacrificed either at mid-light or mid-dark, were similar. The hepatic reductase activity was found to be four- to five-fold greater at mid-dark than at mid-light (462 +/- 141 vs. 123 +/- 22 pmol min-1 mg protein-1). Since the Km value was not modified, the mid-dark activity could be due to an increase in the amount of enzyme. In contrast, HLMC reductase activity and cholesterol content showed similar values at mid-light and mid-dark points. HLMC reductase does not appear to have any diurnal variation and the cholesterol synthesis and content seems to be independent of food intake. HLMC-bearing nude mice undergo several alterations in the biosynthesis and homeostasis of cholesterol. Hypocholesterolemia, lower hepatic cholesterol content and higher HMG-CoA reductase activity are characteristic of host mice.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/metabolismo , Hígado/enzimología , Animales , Carcinoma Mucoepidermoide/metabolismo , Colesterol/análisis , Colesterol/sangre , Femenino , Humanos , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes , Cinética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Microsomas Hepáticos/enzimología , Trasplante de NeoplasiasRESUMEN
Hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase is a highly regulated enzyme which shows a marked circadian rhythmicity. We studied the impact of aging on this rhythm as well as the degree of correlation between age changes in circulating pituitary hormone levels and liver reductase activity in young (4 months) and old (33 months) Sprague-Dawley female rats. Lipid composition was also assessed in plasma and liver microsomes. The maximal activity (midnight) of HMG-CoA reductase fell from 864 +/- 28 pmol mevalonate/min/mg protein in the young rats to 552 +/- 45 pmol/min/mg protein in the old animals, whereas significant change was not observed in the basal (noon) activity levels of the enzyme. Noon serum cholesterol, but not midnight values, was significantly higher in the old rats. Liver cholesterol levels were similar in young and old rats. In old rats, fatty acid composition of liver microsomes revealed an increase in linoleic acid concurrently with a significant decrease in arachidonic acid (AA). A significant correlation was not detected between the age changes in pituitary hormone (GH, PRL, TSH, FSH) serum levels and those in reductase activity. On the other hand, a significant positive correlation was found in the old rats between hepatic reductase activity and the severity of mammary pathology. We conclude that, like most biological rhythms, HMG-CoA reductase circadian fluctuation decreases in amplitude with age. This change does not seem to be linked to the alterations of neuroendocrine function associated with the aging process. The presence of growing mammary tumors seems to stimulate liver reductase activity, which may constitute an adaptive response of the enzyme to cholesterol demand by the growing neoplastic tissue.