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Heterologous expression and characterization of the aspartic endoprotease Pep4um from Ustilago maydis, a homolog of the human Chatepsin D, an important breast cancer therapeutic target

Juárez-Montiel, Margarita, Tesillo-Moreno, Pedro, Cruz-Angeles, Ana, Soberanes-Gutiérrez, Valentina, Chávez-Camarillo, Griselda, Ibarra, J. Antonio, Hernández-Rodríguez, César, Villa-Tanaca, Lourdes
Molecular biology reports 2018 v.45 no.5 pp. 1155-1163
Pichia pastoris, Saccharomyces cerevisiae, Ustilago zeae, active sites, aspartic acid, bioinformatics, breast neoplasms, cathepsin D, enzyme activity, genes, hemoglobin, heterologous gene expression, humans, molecular cloning, pH, recombinant proteins, signal peptide, therapeutics, vacuoles
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.