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Memórias do Instituto Oswaldo Cruz, Vol. 103, No. 6, September, 2008, pp. 598-601 SHORT COMMUNICATION Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress Viviane Giese; Bruno Dallagiovanna; Fabricio K Marchini; Daniela P Pavoni; Marco A Krieger; Samuel Goldenberg+ Instituto Carlos
Chagas-Fiocruz, R. Professor Algacyr Munhoz Mader 3775, 81350-010 Curitiba,
PR, Brasil Financial support: CNPq, PRONEX (Fundação Araucária), PAPES-FIOCRUZ Received 25 March
2008 Code Number: oc08108 ABSTRACT Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi. Key words: calpain-like - Trypanosoma cruzi - stress - metacyclogenesis Understanding the differentiation process of pathogenic protozoa may contribute to the identification of alternative possibilities for developing new drugs and treatment strategies for parasitic diseases. Trypanosoma cruzi is the causative agent of Chagas disease (Chagas 1909). Its life-cycle includes the transformation from non-infective and replicating epimastigotes into non-dividing and infective metacyclic trypomastigotes. This process, referred to as metacyclogenesis, occurs naturally in the gut of the triatomine insect vector and can be reproducibly mimicked in vitro under chemically defined conditions (Contreras et al. 1985). Various cell populations can be distinguished during the in vitro metacyclogenesis process: epimastigotes harvested from growing medium (LIT), epimastigotes subjected to a nutritional stress (NS) (a requirement for differentiation), differentiating epimastigotes (parasites adhered to the culture flask at various times during the process) and metacyclic trypomastigotes (Bonaldo et al. 1988). The transcriptomes of these cell populations determined by microarray analysis have been compared and various differentially expressed genes have thereby been identified. Interestingly, the gene encoding a calpain-like protein (the gene was recently named TcCALPx11) is up-regulated in epimastigotes under NS (Epi-stress) (Probst et al., unpublished observations). Calpains (CA) are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes including apoptosis, signal transduction, differentiation and cytoskeleton remodeling (Ono et al. 1998). Calpain-like proteins (CALP) are atypical CAs and are widespread in lower eukaryotes. Most are devoid of catalytic activity due to amino acid changes within the active-site and/or the absence of EF-hand calcium-binding domains. Nevertheless, these proteins are involved in signal transduction and cell differentiation (Dear et al. 1997). Genome sequences of the Tritryps (T. cruzi, Trypanosoma brucei and Leishmania major) have recently become available: the cysteine peptidases in these species are a large family of proteases which include CALP (Ivens et al. 2005). A recent study provided a comprehensive in silico analysis of CALP in Tritryps and reported their potential role in cell functions (Ersfeld et al. 2005). Here, we describe the characterization of a member of this family, which is differentially expressed during the life-cycle of T. cruzi. The gene encoding the T. cruzi CALP, TcCALPx11 (GeneDB ID Tc00.1047053506563.210), was identified by microarray analysis as being differentially expressed by T. cruzi during metacyclogenesis. Its expression during NS preceding T. cruzi metacyclogenesis was 2.53 times higher than that observed in epimastigotes (Probst et al. unpublished). This gene is a member of group 1, the group consisting of the most conserved CALP (Ersfeld et al. 2005). TcCALPx11 has 692 amino acid residues with an estimated molecular weight of 78.2 kDa, showing 60% amino acid identity with TbCALP1.2 (GeneDB ID Tb927.1.2110) of T. brucei. The active catalytic site of calpain proteases presents an amino acid triad consisting of cysteine, histidine and arginine (Sorimachi et al. 2001). In the case of TcCALPx11, the cysteine is substituted by a serine residue; indeed, amino acid changes in the active site are common in CALP. However, bioinformatics analysis of TcCALPx11 gave no indication of putative acylation motifs, as is the case in the T. brucei related CAP5.5 (Hertz-Fowler et al. 2001), but several phosphorylation sites were identified. We used real-time PCR (qPCR) to quantify Tccalpx11 mRNA in polysomal mRNA fractions (Goldenberg et al. 1985) from epimastigotes, epimastigotes under NS and 24h-adhered (differentiating) parasites; values obtained were normalized to those for the mRNAs encoding GAPDH (Tc00.1047053503687.20), histone H2B (Tc00.1047053511635.10) and a hypothetical protein gene (Tc00.1047053510351.80). Tccalpx11 mRNA was indeed 2.5 times more abundant on average in epimastigotes under NS than in epimastigotes growing in complete medium (Table). There were no major differences in the amounts of the total RNAs, suggesting that stage-specific expression is the result of differential mobilization of mRNAs to the polysome fraction, as described for other T. cruzi genes (Avila et al. 2003). Antiserum against a recombinant TcCALPx11-GST-fusion protein was used to test for TcCALPx11 production. Protein extracts from T. cruzi at various stages of differentiation were analyzed by western blotting and a single 80 kDa protein band was found in epimastigotes. This corresponds to the estimated molecular weight of TcCALPx11 without post-translational acylation, thereby confirming the prediction from in silico analysis. Analysis of protein extracts from parasites during metacyclogenesis showed an increase in the abundance of TcCALPx11 in parasites under NS, whereas it was not detected in metacyclic trypomastigote extracts (Fig. 1A). Furthermore, TcCALPx11 was not detected by western blotting during T. cruzi amastigogenesis (Fig. 1B), suggesting that it is epimastigote-specific. As mentioned previously CA are involved in signal transduction and cytoesqueletal remodeling; the epimastigote-specific expression could suggest a role of TcCALPx11 in the adaptation of this form to the insect vector environment. Proteomic and microarray analysis data suggest that several of the CALP identified in kinetoplastida are differentially expressed (Minning et al. 2003, Atwood et al. 2005). For example, the CAP5.5 protein of T. brucei is differentially expressed during the cell cycle with an increase in the procyclic stage (Hertz-Fowler et al. 2001) and the META 2 protein of Leishmania amazonensis (a SKCRP type protein) is most abundant in promastigotes (Ramos et al. 2004). We analyzed protein extracts prepared from parasites after having undergone various periods of NS and found that the abundance of TcCALPx11 increased two-fold after 1 h of stress (Fig. 1C). This was consistent with the microarray and real-time PCR data, which showed a 2.5-fold increase in the expression of TcCALPx11. This increased expression at the onset of the metacyclogenesis is consistent with a role in the differentiation process; alternatively, TcCALPx11 might be a stress-induced protein. We tested three types of stress (nutritional, temperature and acidic pH) and found that TcCALPx11 expression increased irrespective of the type of stress applied to the parasite (Fig. 1D). Thus, it is possible that TcCALPx11 has a general role in the T. cruzi stress response. To study the function of TcCALPx11, the full-length gene was inserted into the pTEX expression vector and vector was introduced into the parasite. Western blot analysis showed a 3-fold increase of the protein in transfected parasites compared with control parasites (Fig. 2A). However, no morphological or structural alterations were evident in the transfected cells. In addition, over-production of TcCALPx11 did not alter the growth curves or the differentiation rates of the parasites (not shown). In the various organisms in which they have been identified, CA are cytoplasmic proteins commonly associated with the cytoskeleton or cellular membranes through fatty acid modifications (Sorimachi et al. 2001). No potential sites for myristoylation or palmitoylation were found in the aminoacid sequence. The predicted absence of acyl modifications suggests that TcCALPx11 is not associated with membranes. Immunolocalization assays (Fig. 2B) with wild-type and transfected epimastigote forms indicated that TcCALPx11 is in the cytoplasm and is slightly more expressed in transfected parasites (Fig. 2B-1) than in wild-type parasites (Fig. 2B-4). T. brucei CAP5.5 calpain protein was found to be associated with the parasite cytoskeleton. We performed biochemical fractionation of cells into detergent soluble and insoluble fractions as previously described (Woods et al. 1989). Western blot analysis of the corresponding protein fractions showed that the protein partitioned mainly in the insoluble fraction, though some signal could be observed in the soluble fraction. Expressions of ²-tubulin and TcPUF6, a soluble cytoplasmic RNA binding protein (Dallagiovanna et al. 2005), were used as controls of the integrity of protein extracts (Fig. 2C). We also tried to characterize any protease activity of TcCALPx11: we used SDS polyacrylamide gels containing co-polymerized gelatin and in vitro protease assays (d'Avila-Levy et al. 2003) to assay the soluble recombinant fusion protein and the native protein immunoprecipitated from T. cruzi extracts. However, we detected no proteolytic activity, consistent with other CLP. The loss of the protease function and the differential expression observed suggests that this protein may have a role in signal transduction.
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