Characterization of a homologue of SIR2RP1 from Trypanosoma cruzi.

SILVEIRA, R.C.V.; MEYER, C.G.; RAMIREZ, J.C.; CURTO, M.L.A.; ELIAS, M.C.; SCHIJMAN, A.G.; ALONSO G.D.; CANO, M.I.N.

Mar del Plata, Buenos Aires.

Congreso; IX Congreso Argentino de Protozoologia y Enfermedades Parasitarias.; 2011

Sociedad Argentina de Protozoología

Sirtuin proteins belong to a family of NAD+2-dependent protein deacetylases, potential drug targets against many diseases. We present the first characterization of a sirtuin orthologue from Trypanosoma cruzi, the etiologic agent of Chagas disease, the second highest illness burden among neglected tropical diseases. Searching the T. cruzi CL-Brener genome (TriTrypDB) for sirtuin homologues using blastp, we found two Sir2 family proteins, one named TcSIR2RP1 (Esmeraldo like Tc00.1047053508207.150 and Non Esmeraldo like Tc00.1047053507519.60 haplotypes) that shares 56% identity with Sir2RP1 from three different Leishmania species (L. major, L. infantum, L. amazonensis)and the other one (Esmeraldo like Tc00.1047053447255.20 and Non-Esmeraldo like Tc00.1047053506559.80 haplotypes) that shares higher identity (68%) with Sir2RP3 from Leishmania, and thus it could correspond to a TcSIR2RP3 protein. In contrast, a T. cruzi SIR2RP2 orthologue has not been found in the TriTrypDB. In silico analysis showed that both T cruzi Sir2 family like proteins harbor a) a conserved fFGEnl motif that has been shown to be responsible for binding to an acetylated substrate, b) motifs and residues responsible for interaction with NAD+ and deacetylase activity, c) the conserved HG motif that is important for ADP ribosyltransferase activity and d) GAG and NID motifs and the C4 zinc-finger domain, essential for the transcriptional silencing activity present in all Sir2-related proteins. Sir2RP1 from Leishmania sp is secreted and seems to be directly involved in disease pathogenesis since it was found circulating in the bloodstream of dogs infected with L. infantum. In this context, we wanted to characterize TcSir2RP1. Indirect immunofluorescence with antibodies against Sir2RP1 from Leishmania sp recognized a protein in the cytoplasm of CL-Brener epimastigote forms and Western blot analysis depicted a single ~39 kDa polypeptide in whole extracts from k98 (Tc I) and RA (Tc VI) strains, the predicted molecular weight of TcSir2RP1 protein. The cytoplasmatic localization and the conservation of amino acid motifs and residues shared by TcSir2RP1 and Leishmania Sir2RP1 suggest these proteins may also share conserved roles. Moreover, the TcSIR2RP1 Non Esmeraldo like allele was cloned by PCR. Genomic Southern blot analysis allowed identification of a single copy gene and a transcript of less than 1.5 Kb was detected in epimastigotes forms by Northern blot. Expression of the recombinant TcSIR2RP1 protein into a bacterial (pET22b+) and protozoan (pTREX) system is under investigation.