XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:115-2

Poster (Painel)

115-2	CHARACTERIZATION OF Paracoccidioides brasiliensis ANTIZYME BY OVEREXPRESSION IN Saccharomyces cerevisiae
Authors:	Françoise Sorais (IVIC - Instituto Venezolano de Investigaciones Cientificas) ; Martha Cordero (IVIC - Instituto Venezolano de Investigaciones Cientificas) ; Gioconda San-blas (IVIC - Instituto Venezolano de Investigaciones Cientificas) ; Gustavo Nino-vega (IVIC - Instituto Venezolano de Investigaciones Cientificas)

Abstract

Ornithine decarboxylase antizyme, simply called antizyme (OAZ), is a protein that stimulates ornithine decarboxylase (ODC) degradation. This inhibitor of ODC is synthesized in response to an increase in polyamine content via a frameshifting event. It binds to the ODC monomer forming a heterodimer, which prevents enzymatic activity and directs ODC to the proteasome for degradation. Antizyme plays an important role in the regulation of intracellular polyamine levels which are involved in cellular growth and differentiation. In the present study, we tested the functionality of Paracoccidioides brasiliensis antizyme. In this fungus we have identified one antizyme gene (PbrOAZ). The antizyme mRNA contains two overlapping open reading frames (ORFs) comprised of a short ORF1 (170 bp) and a long ORF2 (649 bp). This latter is in the +1 frame relative to ORF1. In order to selectively amplify the complete functional antizyme, which is made up of both ORF1 and ORF2, we used the overlap extension technique. To study overexpression of the P. brasiliensis OAZ gene in a Saccharomyces cerevisiae odc null mutant, we performed a yeast complementation system as described by Mangold and Leberer, 2005. S. cerevisiae odc null mutant (ATCC 4005034) was transformed with pYC2/CT-PbrODC and either pYES2/CT or pYES2/CT-PbrOAZ and absorbance of cultures was measured at 600 nm. As a control, yeast cells were also transformed with both the empty pYC2/CT and pYES2/CT vectors. We show that OAZ can inhibit the growth by interfering with ODC. This result indicate that regulation of P. brasiliensis ODC may be achieved by an unusual post-translational mechanism that possibly involves the antizyme-ODC interaction for the ultimate degradation of the ODC by the proteasome, as it has been described in other organisms. ਀ Financial support: IVIC, project 112.

Keyword: Ornithine decarboxylase, Antizime, Proteasome

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