Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Endocrinology ; 158(11): 3974-3987, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28938463

ABSTRACT

Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterol and steroid biosynthesis that is involved in folliculogenesis and oocyte maturation, which is regulated by follicle-stimulating hormone (FSH), as a key reproductive hormone during follicular development. Thyroid hormone (TH) is also important for normal reproductive function. Although 3,5,3'-triiodothyronine (T3) enhances FSH-induced preantral follicle growth, whether and how TH combines with FSH to regulate CYP51 expression during the preantral to early antral transition stage is unclear. The objective of this study was to determine the cellular and molecular mechanisms by which T3 and FSH regulate CYP51 expression and steroid biosynthesis during preantral follicle growth. Our results indicated that CYP51 expression was upregulated in granulosa cells by FSH, and this response was enhanced by T3. Moreover, knockdown CYP51 decreased cell viability. Meanwhile, gene knockdown also blocked T3 and FSH-induced estradiol (E2) and progesterone (P4) synthesis. These changes were accompanied by upregulation of phospho-GATA-4 content. Results of small interfering RNA analysis showed that knockdown of GATA-4 significantly diminished CYP51 gene expression as well as E2/P4 levels. Furthermore, thyroid hormone receptor ß was necessary to the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), which was required for the regulation of CYP51 expression; activated GATA-4 was also involved these processes. Our data demonstrate that T3 and FSH cotreatment potentiates cellular development and steroid biosynthesis via CYP51 upregulation, which is mediated through the activation of the PI3K/Akt pathway. Meanwhile, activated GATA-4 is also involved in this regulatory system. These findings suggest that CYP51 is a mediator of T3 and FSH-induced follicular development.


Subject(s)
Follicle Stimulating Hormone/pharmacology , Gonadal Steroid Hormones/biosynthesis , Granulosa Cells/drug effects , Ovarian Follicle/drug effects , Sterol 14-Demethylase/physiology , Triiodothyronine/pharmacology , Animals , Animals, Outbred Strains , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Estradiol/metabolism , Female , Granulosa Cells/metabolism , Mice , Oocytes/drug effects , Oocytes/physiology , Ovarian Follicle/physiology , Progesterone/metabolism , Sterol 14-Demethylase/genetics
2.
Rev Argent Microbiol ; 48(2): 137-42, 2016.
Article in English | MEDLINE | ID: mdl-27311753

ABSTRACT

The molecular basis of fluconazole resistance in Cryptococcus neoformans has been poorly studied. A common azole resistance mechanism in Candida species is the acquisition of point mutations in the ERG11 gene encoding the enzyme lanosterol 14-α-demethylase, target of the azole class of drugs. In C. neoformans only two mutations were described in this gene. In order to evaluate other mutations that could be implicated in fluconazole resistance in C. neoformans we studied the genomic sequence of the ERG11 gene in 11 clinical isolates with minimal inhibitory concentration (MIC) values to fluconazole of ≥16µg/ml. The sequencing revealed the G1855A mutation in 3 isolates, resulting in the enzyme amino acid substitution G484S. These strains were isolated from two fluconazole-treated patients. This mutation would not intervene in the susceptibility to itraconazole and voriconazole.


Subject(s)
Antifungal Agents/pharmacology , Cryptococcosis/microbiology , Cryptococcus neoformans/genetics , Drug Resistance, Fungal/genetics , Fluconazole/pharmacology , Fungal Proteins/genetics , Mutation, Missense , Point Mutation , Sterol 14-Demethylase/genetics , Amino Acid Substitution , Antifungal Agents/therapeutic use , Cryptococcosis/drug therapy , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/isolation & purification , Fluconazole/therapeutic use , Fungal Proteins/physiology , Humans , Itraconazole/pharmacology , Meningitis, Cryptococcal/drug therapy , Meningitis, Cryptococcal/microbiology , Microbial Sensitivity Tests , Recurrence , Sterol 14-Demethylase/physiology , Structure-Activity Relationship , Voriconazole/pharmacology
3.
PLoS Negl Trop Dis ; 9(3): e0003588, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25768284

ABSTRACT

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.


Subject(s)
14-alpha Demethylase Inhibitors/pharmacology , Ergosterol/biosynthesis , Leishmania donovani/drug effects , 14-alpha Demethylase Inhibitors/therapeutic use , Animals , Cells, Cultured , Female , Humans , Leishmania donovani/metabolism , Leishmaniasis, Visceral/drug therapy , Mice , Mice, Inbred BALB C , Sterol 14-Demethylase/analysis , Sterol 14-Demethylase/genetics , Sterol 14-Demethylase/physiology
4.
Biochim Biophys Acta ; 1840(6): 1825-36, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24361620

ABSTRACT

BACKGROUND: Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes. METHODS: PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS. RESULTS: Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26µM for ketoconazole and 0.64µM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s. CONCLUSIONS: Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s. GENERAL SIGNIFICANCE: The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.


Subject(s)
Recombinant Proteins/chemistry , Sterol 14-Demethylase/chemistry , Animals , Female , Humans , Ligands , Male , Models, Molecular , Molecular Docking Simulation , Sterol 14-Demethylase/physiology , Sterols/biosynthesis , Zebrafish
5.
Rev Iberoam Micol ; 30(3): 209-12, 2013.
Article in English | MEDLINE | ID: mdl-23402828

ABSTRACT

BACKGROUND: For many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species. AIMS: In this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole. METHODS: Screening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing. RESULTS: We identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p. CONCLUSION: The L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.


Subject(s)
Candida albicans/genetics , Drug Resistance, Fungal/genetics , Fluconazole/pharmacology , Fungal Proteins/genetics , Genes, Fungal , Mutation, Missense , Point Mutation , Sterol 14-Demethylase/genetics , Alleles , Candida albicans/drug effects , Candida glabrata/genetics , Candida tropicalis/genetics , Candidemia/microbiology , Candidiasis, Oral/microbiology , DNA Mutational Analysis , DNA, Fungal/genetics , Fungal Proteins/chemistry , Fungal Proteins/physiology , Humans , Models, Molecular , Protein Conformation , Sterol 14-Demethylase/chemistry , Sterol 14-Demethylase/physiology
6.
J Mycol Med ; 22(2): 173-8, 2012 Jun.
Article in English | MEDLINE | ID: mdl-23518020

ABSTRACT

The molecular mechanisms supporting resistance to azole antifungals have attracted a great interest during the last decades because of the emergence of clinical resistance to the treatment of fungal infections. The availability of genome sequencing data, of molecular biology tools, and of a large set of clinical and laboratory azole-resistant strains, made the yeasts Candida the biological material of choice to decipher azole resistance mechanisms. The yeast Candida albicans has several cellular ways to resist to azole drugs: decreased affinity of the target protein Erg11p for the drugs, increased biosynthesis of Erg11p, and efflux of the drugs outside the fungal cells. At the molecular level, two main mechanisms are operating: point mutation in the target gene or in transcriptional activator factors, eventually associated to a loss of heterozygosity, and gene duplication that results from the extraordinary plasticity of the genome. This review proposes to explore the different molecular strategies that are used by Candida yeasts to fight azole antifungals.


Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Drug Resistance, Multiple, Fungal/physiology , Fungal Proteins/physiology , Sterol 14-Demethylase/physiology , Transcription Factors/physiology , Triazoles/pharmacology , ATP-Binding Cassette Transporters/physiology , Amino Acid Motifs , Aneuploidy , Biological Transport , Candida/enzymology , Candida/genetics , Candida/physiology , Chromosomes, Fungal/genetics , Drug Resistance, Multiple, Fungal/genetics , Endoplasmic Reticulum, Smooth/enzymology , Ergosterol/metabolism , Fungal Proteins/genetics , Gene Duplication , Gene Expression Regulation, Fungal , Genes, Fungal , Haploidy , Isochromosomes/genetics , Membrane Lipids/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/physiology , Point Mutation , Protein Binding , Sterol 14-Demethylase/genetics , Trans-Activators/physiology , Transcription Factors/genetics , Up-Regulation
7.
Drug Metab Dispos ; 39(6): 966-73, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21368239

ABSTRACT

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.


Subject(s)
Lipidoses/enzymology , Liver/enzymology , NADPH-Ferrihemoprotein Reductase/antagonists & inhibitors , Animals , Cell Line, Tumor , Gas Chromatography-Mass Spectrometry , Immunoblotting , Lipid Metabolism/physiology , Lipidoses/genetics , Lipidoses/metabolism , Liver/metabolism , Mice , Mice, Knockout , Microscopy, Fluorescence , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/physiology , Plasmids , RNA, Small Interfering/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sterol 14-Demethylase/genetics , Sterol 14-Demethylase/metabolism , Sterol 14-Demethylase/physiology , Transfection , Triglycerides/genetics , Triglycerides/metabolism
8.
Pest Manag Sci ; 67(1): 44-59, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20949586

ABSTRACT

BACKGROUND: Sterol 14α-demethylation inhibitors (DMIs) have been widely used in many European countries to control septoria leaf blotch, which is caused by Mycosphaerella graminicola (Fückel) J Schrot (anamorph Septoria tritici Berk & MA Curtis). However, treatment efficacy has declined, and significant shifts in population susceptibility have occurred in recent years, with the isolation of particularly highly resistant strains from French, English and Irish populations. The present aim was to determine the phenotypic characteristics of these field isolates and to identify the possible resistance mechanisms. RESULTS: Target alteration, linked to 11 possible changes in the gene encoding 14α-demethylase (Cyp51), was the basic resistance mechanism in weakly, moderately and highly resistant strains. Changes in Cyp51 combined with the overexpression of drug efflux transporters probably result in multidrug resistance in some of the most resistant phenotypes. Finally, some moderately or highly resistant isolates were found to harbour an insertion in the Cyp51 promoter and/or new combinations of known mutations in the target gene. CONCLUSION: An updated overview of M. graminicola field strains displaying low to high resistance to DMIs is provided here. The management of field resistance and efficacy should be adapted to take these findings into account.


Subject(s)
14-alpha Demethylase Inhibitors/pharmacology , Ascomycota/drug effects , Fungicides, Industrial/pharmacology , 14-alpha Demethylase Inhibitors/chemistry , Amino Acid Sequence , Ascomycota/isolation & purification , Drug Resistance, Fungal , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Fungal Proteins/physiology , Fungicides, Industrial/chemistry , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/physiology , Molecular Sequence Data , Pest Control , Sequence Alignment , Sterol 14-Demethylase/chemistry , Sterol 14-Demethylase/physiology
SELECTION OF CITATIONS
SEARCH DETAIL
...