RESUMO
BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Proteínas Recombinantes de Fusão/farmacologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/uso terapêutico , Especificidade de Anticorpos/imunologia , Antígenos de Protozoários/imunologia , Linhagem Celular , Clonagem Molecular , Reações Cruzadas/imunologia , Desenvolvimento de Medicamentos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Imunofluorescência , Expressão Gênica , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Mimetismo Molecular , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Análise de Sequência de DNA , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
NKX2-5 is a homeodomain transcription factor that plays a crucial role in heart development. It is the first gene where a single genetic variant (GV) was found to be associated with congenital heart diseases in humans. In this study, we carried out a comprehensive survey of NKX2-5 GVs to build a unified, curated, and updated compilation of all available GVs. We retrieved a total of 1,380 unique GVs. From these, 970 had information on their frequency in the general population and 143 have been linked to pathogenic phenotypes in humans. In vitro effect was ascertained for 38 GVs. The homeodomain had the biggest cluster of pathogenic variants in the protein: 49 GVs in 60 residues, 23 in its third α-helix, where 11 missense variants may affect protein-DNA interaction or the hydrophobic core. We also pinpointed the likely location of pathogenic GVs in four linear motifs. These analyses allowed us to assign a putative explanation for the effect of 90 GVs. This study pointed to reliable pathogenicity for GVs in helix 3 of the homeodomain and may broaden the scope of functional and structural studies that can be done to better understand the effect of GVs in NKX2-5 function.
Assuntos
Proteína Homeobox Nkx-2.5/genética , Motivos de Aminoácidos , Bases de Dados Genéticas , Humanos , Mutação , Estrutura Secundária de ProteínaRESUMO
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroidogenesis. Disorders in steroid 21-hydroxylation account for over 95% of patients with CAH. Clinically, the 21-hydroxylase deficiency has been classified in a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non-classical. Known allelic variants in the disease causing CYP21A2 gene are spread among different sources. Until recently, most variants reported have been identified in the clinical setting, which presumably bias described variants to pathogenic ones, as those found in the CYPAlleles database. Nevertheless, a large number of variants are being described in massive genome projects, many of which are found in dbSNP, but lack functional implications and/or their phenotypic effect. In this work, we gathered a total of 1,340 GVs in the CYP21A2 gene, from which 899 variants were unique and 230 have an effect on human health, and compiled all this information in an integrated database. We also connected CYP21A2 sequence information to phenotypic effects for all available mutations, including double mutants in cis. Data compiled in the present work could help physicians in the genetic counseling of families affected with 21-hydroxylase deficiency.
Assuntos
Bases de Dados Genéticas , Variação Genética , Mutação , Esteroide 21-Hidroxilase/genética , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Alelos , Estudos de Associação Genética , Genótipo , Humanos , FenótipoRESUMO
Enzyme catalysis was applied to synthesize derivatives of three bile acids and their biological activity was evaluated as growth inhibitors of the protozoan Trypanosoma cruzi. Twelve mono-, diacetyl and ester derivatives of deoxycholic, chenodeoxycholic and lithocholic acid, seven of them new compounds, were obtained through lipase-catalyzed acetylation, esterification and alcoholysis reactions in very good to excellent yield and a highly regioselective way. Among them, acetylated ester products, in which the lipase catalyzed both reactions in one-pot, were obtained. The influence of various reaction parameters in the enzymatic reactions, such as enzyme source, acylating agent/substrate ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evaluated compounds showed a remarkable activity as Trypanosoma cruzi growth inhibitors, obtaining the best results with ethyl chenodeoxycholate 3-acetate and chenodeoxycholic acid 3,7-diacetate, which showed IC50: 8.6 and 22.8 µM, respectively. In addition, in order to shed light to bile acids behavior in enzymatic reactions, molecular modeling was applied to some derivatives. The advantages showed by the enzymatic methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to synthesize these bile acid derivatives with application as potential antiparasitic agents.
Assuntos
Antiprotozoários/farmacologia , Ácidos e Sais Biliares/química , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Acetilação , Antiprotozoários/química , Antiprotozoários/metabolismo , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/farmacologia , Sítios de Ligação , Biocatálise , Avaliação Pré-Clínica de Medicamentos , Esterificação , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Solventes/química , Estereoisomerismo , Especificidade por Substrato , Temperatura , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The rise in the world demand for food poses a challenge to our ability to sustain soil fertility and sustainability. The increasing use of no-till agriculture, adopted in many areas of the world as an alternative to conventional farming, may contribute to reduce the erosion of soils and the increase in the soil carbon pool. However, the advantages of no-till agriculture are jeopardized when its use is linked to the expansion of crop monoculture. The aim of this study was to survey bacterial communities to find indicators of soil quality related to contrasting agriculture management in soils under no-till farming. Four sites in production agriculture, with different soil properties, situated across a west-east transect in the most productive region in the Argentinean pampas, were taken as the basis for replication. Working definitions of Good no-till Agricultural Practices (GAP) and Poor no-till Agricultural Practices (PAP) were adopted for two distinct scenarios in terms of crop rotation, fertilization, agrochemicals use and pest control. Non-cultivated soils nearby the agricultural sites were taken as additional control treatments. Tag-encoded pyrosequencing was used to deeply sample the 16S rRNA gene from bacteria residing in soils corresponding to the three treatments at the four locations. Although bacterial communities as a whole appeared to be structured chiefly by a marked biogeographic provincialism, the distribution of a few taxa was shaped as well by environmental conditions related to agricultural management practices. A statistically supported approach was used to define candidates for management-indicator organisms, subsequently validated using quantitative PCR. We suggest that the ratio between the normalized abundance of a selected group of bacteria within the GP1 group of the phylum Acidobacteria and the genus Rubellimicrobium of the Alphaproteobacteria may serve as a potential management-indicator to discriminate between sustainable vs. non-sustainable agricultural practices in the Pampa region.
Assuntos
Agricultura/métodos , Bactérias/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Microbiologia do Solo , Solo/química , Argentina , Geografia , Filogenia , Solo/normasRESUMO
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 P2ß protein (TcP2ß) 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.
Assuntos
Anticorpos Antiprotozoários/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Protozoários/biossíntese , Proteínas Ribossômicas/antagonistas & inibidores , Anticorpos de Cadeia Única/farmacologia , Trypanosoma cruzi/metabolismo , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/genética , Doença de Chagas/tratamento farmacológico , Doença de Chagas/metabolismo , Epitopos/química , Epitopos/imunologia , Expressão Gênica , Humanos , Modelos Moleculares , Filogenia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/classificação , Proteínas Ribossômicas/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1ß, TcP2α, TcP2ß and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1ß-TcP2α and TcP1ß-TcP2ß. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2ß were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.