Analysis of Proteasomal Proteolysis during the In Vitro Metacyclogenesis of Trypanosoma cruzi

Josiane Cardoso,Daniela F Gradia,Stênio P Fragoso,Renata Guerra De Sá,Marco A Krieger,Carla De Paula Lima,Tiago Leal,Samuel Goldenberg

doi:10.1371/journal.pone.0021027

Josiane Cardoso, Daniela F Gradia + Show 6 more

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https://doi.org/10.1371/journal.pone.0021027

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Abstract

Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell.

Highlights

Proteasomes are the principal non-lysosomal degradation machinery present in all types of eukaryotic cells [1,2]
Specific antibodies directed against the catalytic subunit alpha 7 of the 20S proteasome, the regulatory subunit 10 (RPN10) of 19 S and the proteasome activator 26 (PA26) were used in western blot assays with protein extracts collected during metacyclogenesis
Immunolocalization of alpha 7, RPN10 and PA26 To evaluate the intracellular distribution of proteasomes in parasites we used specific antibodies directed against the catalytic subunit alpha 7 of the 20S proteasome, regulatory subunit 10 (RPN10) of 19 S and proteasome activator 26 (PA26) in epimastigotes (Figure 2A) and metacyclic trypomastigotes (Figure 2B)

Summary

Introduction

Proteasomes are the principal non-lysosomal degradation machinery present in all types of eukaryotic cells [1,2]. The 20S proteasome is the catalytic core of this degradation machinery It is present in a latent form in the cell, but it is activated by various types of regulatory complexes [7]. Proteasome subtypes with different proteolytic properties are formed by the attachment of these regulatory complexes to one or both endplates of the barrel-shaped 20S core particles [8]. This proteasome is a threonine protease with its active sites located within the particle. The physiological function of the 20S proteasome has not been clearly defined, but it is well documented that it degrades oxidized proteins independently of ATP and ubiquitin [10,11,12,13,14]

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