A polyclonal antibody against TcPuf6 (12 μL) was used as a control (α-TcPuf6). The presence/absence selleck of the antibodies and protein extract in the binding reactions is indicated by +/- signs above each lane. Given the proposed roles in telomere and kinetoplast DNA recognition of Tc38 trypanosomatid orthologues, we analyzed whether endogenous Tc38 could also interact with single stranded [dT-dG] rich cis-acting sequences from nuclear and mitochondrial origins. Oligonucleotides containing the sequence of the telomere repeat, a [dT-dG] rich region of the T. cruzi maxicircle that is synthenically located
to the replication origin mapped in T. brucei and the minicircle UMS were assayed in vitro by EMSA with whole T. cruzi epimastigote protein extracts. We observed a pattern of bands similar to that observed for the poly [dT-dG] probe (Figure 1) and these complexes were all supershifted by the anti-Tc38 AUY-922 nmr antibody. Control reactions using the anti-TcPuf6 antibody [24] at the same concentration were unable to produce any supershift. These data suggest that native Tc38 is able
to recognize single stranded [dT-dG] enriched sequences in different contexts and support a possible telomeric or kinetoplast-associated role. Tc38 is expressed throughout T. cruzi life cycle In order to better understand the Tc38 physiological role, we looked at its expression in both proliferative (epimastigotes and amastigotes) and non-proliferative (metacyclic trypomastigotes) stages of the parasite. The polyclonal antiserum raised against GST-Tc38 was used to probe membranes with total protein extracts from different stages by western analysis. As shown in figure 2, a band of 38 kDa was observed in all extracts from the various parasite life cycle stages. Normalization
of Tc38 levels was performed using TcPuf6, another RNA binding protein, which showed minimal variation during T. cruzi life cycle [24]. Figure Diflunisal 2 Expression of Tc38 during the T. cruzi life cycle. Western analysis of total protein extract using purified anti-Tc38 and anti-TcPuf6 antibody is shown. Protein extracts from 1 × 107 parasites were loaded into each lane. Life cycle stages are indicated as: E: epimastigotes, M: metacyclic trypomastigotes and A: amastigotes. Tc38 is found in the T. cruzi mitochondrion Tc38 bears a hypothetical N-terminal mitochondrial targeting signal and its orthologous genes in T. brucei and L. tarentolae have been proposed to encode mitochondrial proteins [11]. TbRBP38/p38 has also been shown to co-localize with the kinetoplast in a T. brucei transfectant overexpressing the fusion protein p38-GFP [10]. However, other researchers have isolated orthologues from a L. amazonensis nuclear enriched fraction and/or for its affinity for nuclear DNA targets [13]. These data together with Tc38 ability to bind kinetoplastid and telomeric sequences could be integrated by proposing a dual localization of this protein, both in the mitochondrion and the nucleus.
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