The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role infungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was over-expressed in bacteria and a polyclonal antibody to this protein was produced. Weidentified a 180-kDa protein species reactive to the antibody produced in miceagainst the P. brasiliensis recombinant purified formamidase of 45 kDa. The180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide massfinger printing using MS. The identical mass spectra generated by the 180 and the45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formami-dase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights intoformamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism

Computational prediction of protein-protein interactions in Leishmania predicted proteomes

Submitted by Nuzia Santos ([email protected]) on 2014-06-26T12:02:30Z No. of bitstreams: 1 Computational prediction of protein-protein interactions in Leishmania predicted proteomes.pdf: 1144181 bytes, checksum: 8031f5a087f544239fbb4964d3259fb1 (MD5)Made available in DSpace on 2014-06-26T12:02:30Z (GMT). No. of bitstreams: 1 Computational prediction of protein-protein interactions in Leishmania predicted proteomes.pdf: 1144181 bytes, checksum: 8031f5a087f544239fbb4964d3259fb1 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrazilFundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Laboratório de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brazil/Universidade Federal de Ouro Preto. Laboratório de Pesquisas Clínicas. Ouro Preto, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Laboratório de Parasitologia Celular e MolecularThe Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI) study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping) and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks received some degree of functional annotation which represents an important contribution since approximately 60% of Leishmania predicted proteomes has no predicted function

Eukaryotic protein kinases (ePKs) of the helminth parasite Schistosoma mansoni.

Submitted by Nuzia Santos ([email protected]) on 2014-07-31T11:09:14Z No. of bitstreams: 1 Eukaryotic protein kinases (ePKs) of the helminth parasite Schistosoma mansoni.pdf: 4374199 bytes, checksum: 9dc18529787c603ef883bdb239790003 (MD5)Made available in DSpace on 2014-07-31T11:09:14Z (GMT). No. of bitstreams: 1 Eukaryotic protein kinases (ePKs) of the helminth parasite Schistosoma mansoni.pdf: 4374199 bytes, checksum: 9dc18529787c603ef883bdb239790003 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Genomics and Computational Biology Group. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Genomics and Computational Biology Group. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Excelência em Bioinformática. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Genomics and Computational Biology Group. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Genomics and Computational Biology Group. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Excelência em Bioinformática. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Excelência em Bioinformática. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Genomics and Computational Biology Group. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Excelência em Bioinformática. Belo Horizonte, MG, BrazilBACKGROUND: Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The Schistosoma mansoni genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the S. mansoni predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets. RESULTS: We have identified 252 ePKs, which corresponds to 1.9% of the S. mansoni predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that S. mansoni has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in S. mansoni or belong to an expanded family in this parasite. Only 16 S. mansoni ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite. CONCLUSIONS: Our approach has improved the functional annotation of 40% of S. mansoni ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of S. mansoni in response to diverse environments during the parasite development, vector interaction, and host infection

Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.

Submitted by Nuzia Santos ([email protected]) on 2014-06-27T15:55:19Z No. of bitstreams: 1 Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.pdf: 3191498 bytes, checksum: 460444e66a425185e6e0d1f7c3fcd48e (MD5)Made available in DSpace on 2014-06-27T15:55:19Z (GMT). No. of bitstreams: 1 Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.pdf: 3191498 bytes, checksum: 460444e66a425185e6e0d1f7c3fcd48e (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil / Fundação Oswaldo Cruz. Centro de Excelência em Bioinformática. Belo Horizonte, MG, Brazil/ Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilBackground: Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS). Methods: In the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms. Results: In silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level. Conclusion: The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressur

The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role infungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was over-expressed in bacteria and a polyclonal antibody to this protein was produced. Weidentified a 180-kDa protein species reactive to the antibody produced in miceagainst the P. brasiliensis recombinant purified formamidase of 45 kDa. The180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide massfinger printing using MS. The identical mass spectra generated by the 180 and the45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formami-dase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights intoformamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide