XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:38-1

Poster (Painel)

38-1	Development of a probe for detection of Ras activated form of Paracoccidioides brasiliensis
Authors:	Ana Eliza Coronel Janu Haniu (UNIFESP - Universidade Federal de São Paulo) ; Rodrigo Bernardi Miguel (UNIFESP - Universidade Federal de São Paulo) ; Hugo Pequeno Monteiro (UNIFESP - Universidade Federal de São Paulo) ; Rosana Puccia (UNIFESP - Universidade Federal de São Paulo) ; Wagner Luiz Batista (UNIFESP - Universidade Federal de São Paulo)

Abstract

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The fungus occurs as two morphological stages, mycelium and yeast. Pathogenic fungi must be able to adapt to dramatic changes as they move from the environment into the infected host. The signaling pathways that are used to detect changes in the environment may also be used to regulate the determinants of virulence and host infection. Ras proteins are important determinants of cell morphology, differentiation, and stress response in diverse organisms. They function like molecular switches cycling between GTP bound “on” and GDP bound “off” states. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic, Byr2 kinase represents a Ras target considered a homolog of mammalian Raf-kinase. In P. brasiliensis, the BYR2 encodes a protein of 894 amino acids. The region that comprises residues 247 to 397 of BYR2 is necessary and sufficient to interact with Ras. In order to characterize the activity function of Ras, we constructed a probe to analysis the Ras activated form. The region of Ras Binding Domain (RBD) was amplified from BYR2 gene by PCR using primers added of the restriction enzymes sites. The GST-RBD fusion protein was prepared using the expression vector pGEX4T2-RBD, which encodes amino acids 247–397 of Byr2 fused to GST. The expression of GST-RBD fusion protein in Escherichia coli (BL21) was induced with 0.1 mM IPTG for 4 h at 30 °C, and the fusion protein was purified on glutathione-Sepharose beads. In parallel we have investigated the possibility of using the probe RBD-GST (Raf-1 human) to detect the activated form of Ras in P. brasiliensis. In this work we cloned, expressed and constructed a probe from RBD of BYR2 gene of P. brasiliensis. The probe will be used to detect Ras activated form of fungus. Financial support: CNPq, CAPES.

Keyword: BRY2, Ras GTPase, RBD-GST probe

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