Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression

• Published in: Revista iberoamericana de micología Vol. 33; no. 1; pp. 26 - 33
• Main Authors: Sepulveda-Gonzalez, Ma Eugenia, Parra-Ortega, Berenice, Betancourt-Cervantes, Yuliana, Hernandez-Rodriguez, Cesar, Xicohtencatl-Cortes, Juan, Villa-Tanaca, Lourdes
• Format: Journal Article
• Language: Spanish
• Published: 01.03.2016
• Subjects: Candida glabrata; Saccharomyces cerevisiae
• ISSN: 1130-1406
• DOI: 10.1016/j.riam.2014.10.005

Background: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen. Resumen Antecedentes La vacuola de Saccharomyces cerevisiae esta involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradacion, recambio proteico, desintoxicacion y proteccion de la celula en condiciones de estres. Por el contrario, las proteasas vacuolares de Candida glabrata aun no han sido estudiadas por completo. Objetivos El presente trabajo describe por primera vez la actividad proteolitica vacuolar en C. glabrata. Metodos Los estudios bioquimicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteoliticas: aspartil proteinasa acida, que actua sobre sustratos como la albumina y la hemoglobina acida desnaturalizada; serin proteasa neutra, con actividad sobre el substrato de tipo colageno hide powder azure, y serin carboxipeptidasa, que actua sobre N-benzoil-tyr-pNa. Resultados La obtencion de una fraccion subcelular mostro una elevada actividad enzimatica especifica de las tres proteasas, lo que permitio confirmar su localizacion vacuolar. Se realizaron analisis de la expresion de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteoliticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulacion diferencial de la expresion de la proteasa, dependiendo de la fuente de nitrogeno. Conclusiones Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrian participar en el proceso de autofagia y supervivencia de este patogeno oportunista.