Biochemical characterization of TcCYC6, a protein with cyclin homology from Trypanosoma cruzi

Abstract

The cell cycle is a highly regulated process that requiresCyclin-Dependent Kinases (CDKs) in association with cyclins and other factors to activate orinactivate biochemical pathways that leads to proper duplication and segregationinto new daughter cells. Which are these regulators controlling the cell cyclein Trypanosoma cruzi, a pathogenparasite that alters between proliferative and non dividing forms, is stillpoorly understood and deserves further studies. The genome of T. cruzi codify for ten sequences withcyclin homology (TcCYC2 to TcCYC11), three of which werecharacterized at the functional level by our group (TcCYC2, TcCCY4 and TcCYC5). In order to further understandthe role of the cyclin family in the cell cycle control of this parasite, a sequencewith similarity to mitotic cyclins (TcCYC6),was studied. For this purpose, we first raised polyclonal antibodies against theN-terminal peptide of TcCYC6 protein.This antibody was unable to detect the endogenous expression of TcCYC6 in epimastigotes either bywestern blot or immunofluorescence microscopy. However, we isolated the TcCYC6 messenger RNA, indicating that thisgene is processed at least as mature transcript. Then, we cloned the TcCYC6 coding sequence fused to the influenzahemagglutinin tag, HA (TcCYC6-HA)into the inducible expression vector pTcINDEX.Over-expression of TcCYC6-HA causesinhibition of epimastigote growth, although the cell cycle pattern of parasitepopulation is not compromise. In agreement with this, studies usingimmunofluorescence microscopy did not reveal any cell cycle dependentre-localization of this recombinant protein. However, results from treatmentwith specific inhibitors and affinity chromatography suggested that TcCYC6-HA protein could be degraded byproteasome pathway. Complementation assays suggested that TcCYC6-HA does notcomplement the G1 cyclin deficiency in yeasts, although other functionalexperiments are in progress. Supported byPICT 2012; CONICET