RESUMO
The pep4um gene (um04926) of Ustilago maydis encodes a protein related to either vacuolar or lysosomal aspartic proteases. Bioinformatic analysis of the Pep4um protein revealed that it is a soluble protein with a signal peptide suggesting that it likely passes through the secretory pathway, and it has two probable self-activation sites, which are similar to those in Saccharomyces cerevisiae PrA. Moreover, the active site of the Pep4um has the two characteristic aspartic acid residues of aspartyl proteases. The pep4um gene was cloned, expressed in Pichia pastoris and a 54 kDa recombinant protein was observed. Pep4um-rec was confirmed to be an aspartic protease by specifically inhibiting its enzymatic activity with pepstatin A. Pep4um-rec enzymatic activity on acidic hemoglobin was optimal at pH 4.0 and at 40 °C. To the best of our knowledge this is the first report about the heterologous expression of an aspartic protease from a basidiomycete. An in-depth in silico analysis suggests that Pep4um is homolog of the human cathepsin D protein. Thus, the Pep4um-rec protein may be used to test inhibitors of human cathepsin D, an important breast cancer therapeutic target.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Clonagem Molecular/métodos , Ustilago/enzimologia , Ácido Aspártico Endopeptidases/metabolismo , Domínio Catalítico , Catepsina D/genética , Simulação por Computador , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Peso Molecular , Filogenia , Pichia/genética , Pichia/crescimento & desenvolvimento , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ustilago/genéticaRESUMO
The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k m and V max for the HMG-CoA were 6.5 µM and 2.26 × 10-3 µM min-1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5 µM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.
Assuntos
Candida glabrata/enzimologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Terapia de Alvo Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Biocatálise , Candida glabrata/genética , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Cinética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteínas Recombinantes/química , Sinvastatina/farmacologia , Solubilidade , Especificidade por Substrato , TemperaturaRESUMO
The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).