The Ubiquitin−Proteasome Pathway Plays an Essential Role in Proteolysis during Trypanosoma cruzi Remodeling

Abstract

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

“…The subsequent morphological changes involving thickening of the posterior end and progressive shortening of the parasite around itself in a helical fashion, along with a gradual internalization of the flagellum resemble the changes for tissue-culture-derived trypomastigotes as described above. The fact that early and late morphological changes are associated with the different environmental conditions surrounding extracelullar primary amastigogenesis may provide an opportunity to study the role of ubiquitin (UB)-proteosome system, which was recently shown to be involved in the remodeling process of trypomastigote transformation (De Diego et al 2001). It is worth noting that the early events described during primary amastigogenesis were not observed when tissueculture derived trypomastigotes transformed into amastigotes.…”

Antileishmanial activity of HIV protease inhibitors

“…Proteasomes are large, non-lysosomal, multi-subunit protease complexes. They are characterised by evolutionarily conserved proteins and are present in Trypanosoma brucei [8], Trypanosoma cruzi [9], Toxoplasma gondii [10], Plasmodium spp. [11], Entamoeba histolytica and Entamoeba invadens [12], as well as in Leishmania mexicana [13].…”

Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development

“…De Diego et al (2001) assumed that the enzyme plays an important role during the remodeling phase of Trypanosoma cruzi and indicated a significant role of the proteasome during the development of organisms. In the arthropod Drosophila melanogaster, a cell-specific accumulation of proteasome was reported during the early development (Klein et al 1990).…”

“…We assume that this mechanism, as it represents a period of enhanced protein turnover, will strongly depend on the balanced interplay of proteasomal activities. The proteasome is one of the main regulators of the cell-cycle (Richter-Ruoff and Wolf 1993;Naujokat and Hoffmann 2002), differentiation processes such as remodeling (De Diego et al 2001) and growth. Therefore, it seems likely that especially in the developmental stages which are characterized by high mitotic activity, growth and high transcription rates might demand an active and regulating proteasome.…”