Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein (original) (raw)
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The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2b protein (TcP2b) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.
Proteomic analysis of the Trypanosoma cruzi ribosomal proteins
Biochemical and Biophysical Research Communications, 2009
Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have $50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N-or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.
Parasitology, 2003
Small nuclear ribonucleoproteins (snRNPs) are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-S...
PMC Biophysics, 2009
The acidic C-terminal peptides from Trypanosoma cruzi ribosomal P proteins are the major target of the antibody response in patients suffering Chagas chronic heart disease. It has been proposed that the disease is triggered by the cross-reaction of these antibodies with the second extra cellular loop of the 1-adrenoreceptor, brought about by the molecular mimicry between the acidic C-terminal peptides and the receptor's loop. To improve the understanding of the structural basis of the autoimmune response against heart receptors, the 3-dimensional structure of the C-terminal peptides of Trypanosoma cruzi ribosomal proteins P0 (EDDDDDFGMGALF) and P2 (EEEDDDMGFGLFD) were solved using the Electrostaticaly Driven MonteCarlo method. Their structures were compared with the second extra-cellular loop of our homology model of human rhodopsin and the existing experimental NMR structures of the C-terminal peptides from human P0 (EESDDDMGFGLFD) and from Leishmania braziliensis P0 (EEADDDMGFGLFD). Docking of Trypanosoma cruzi peptides P0, P2 and human rhodopsin loop into our anti-P2 monoclonal antibody homology model allowed to explore their interactions.
Microbes and Infection, 2008
Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.
Protein & Peptide Letters, 2005
The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 Cterminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N-terminal domain belonging to the α/β class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.
The deduced primary structure of a ribosomal protein S4 from Trypanosoma cruzi
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1998
Two cDNA clones encoding a protein homologous to ribosomal protein S4 from the protozoan parasite Trypanosoma cruzi were isolated and characterized. Both clones potentially encode for an identical basic protein of 273 amino acids. Sequence comparisons with other species indicate that this protein is highly conserved. Hybridization studies are consistent with the occurrence of two genes in T. cruzi encoding this ribosomal protein. An RNA of approximately 1 kb was present in both exponentially growing and stationary phase derived epimastigotes, and no significant differences were detected in its steady state concentration.
Protein expression and purification, 2001
The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. How...