RESUMO
During the last decade the number of characterized F420-dependent enzymes has significantly increased. Many of these deazaflavoproteins share a TIM-barrel fold and are structurally related to FMN-dependent luciferases and monooxygenases. In this work, we traced the origin and evolutionary history of the F420-dependent enzymes within the luciferase-like superfamily. By a thorough phylogenetic analysis we inferred that the F420-dependent enzymes emerged from a FMN-dependent common ancestor. Furthermore, the data show that during evolution, the family of deazaflavoproteins split into two well-defined groups of enzymes: the F420-dependent dehydrogenases and the F420-dependent reductases. By such event, the dehydrogenases specialized in generating the reduced deazaflavin cofactor, while the reductases employ the reduced F420 for catalysis. Particularly, we focused on investigating the dehydrogenase subfamily and demonstrated that this group diversified into three types of dehydrogenases: the already known F420-dependent glucose-6-phosphate dehydrogenases, the F420-dependent alcohol dehydrogenases, and the sugar-6-phosphate dehydrogenases that were identified in this study. By reconstructing and experimentally characterizing ancestral and extant representatives of F420-dependent dehydrogenases, their biochemical properties were investigated and compared. We propose an evolutionary path for the emergence and diversification of the TIM-barrel fold F420-dependent dehydrogenases subfamily.
Assuntos
Archaea/enzimologia , Proteínas Arqueais/classificação , Bactérias/enzimologia , Proteínas de Bactérias/classificação , Evolução Molecular , Oxirredutases/classificação , Riboflavina/análogos & derivados , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Escherichia coli/genética , Oxirredutases/química , Oxirredutases/genética , Filogenia , Riboflavina/química , Especificidade por SubstratoRESUMO
Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosidases that inhibit protein synthesis through the elimination of a single adenine residue from 28S rRNA. Many of these toxins have been characterized in depth from a biochemical and molecular point of view. In addition, their potential use in medicine as highly selective toxins is being explored. In contrast, the evolutionary history of RIP encoding genes has remained traditionally underexplored. In recent years, accumulation of large genomic data has fueled research on this issue and revealed unexpected information about the origin and evolution of RIP toxins. In this review we summarize the current evidence available on the occurrence of different evolutionary mechanisms (gene duplication and losses, horizontal gene transfer, synthesis de novo and domain combination) involved in the evolution of the RIP gene family. Finally, we propose a revised nomenclature for RIP genes based on their evolutionary history.
Assuntos
Evolução Molecular , Proteínas Inativadoras de Ribossomos/genética , Animais , Bactérias , Fungos , Duplicação Gênica , Transferência Genética Horizontal , Filogenia , PlantasRESUMO
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
Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by depurinating an adenine on the sarcin-ricin loop (SRL) of the large subunit ribosomal RNA. Several RIPs interact with the C-terminal end of ribosomal stalk P proteins, and this interaction is required for their full activity. In contrast, the activity of Pokeweed Antiviral Protein is not affected by blocking this stalk component. Here, we provide evidence from phylogenetic analyses and sequence alignments suggesting that the interaction with the C-terminal end of P proteins evolved independently in different RIPs by convergent evolution.
Assuntos
Evolução Molecular , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Teorema de Bayes , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Biossíntese de Proteínas , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ricina/genética , Ricina/metabolismo , Alinhamento de SequênciaRESUMO
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.
Assuntos
Complexos Multiproteicos/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/metabolismo , Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Genes de Protozoários , Cinética , Dados de Sequência Molecular , Complexos Multiproteicos/química , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas Ribossômicas/química , Análise de Sequência de Proteína , Ressonância de Plasmônio de Superfície , Trypanosoma cruzi/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
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 approximately 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.
Assuntos
Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Proteômica , Proteínas Ribossômicas/genética , Trypanosoma cruzi/genéticaRESUMO
Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sdelta. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A(51) residue on 28Sdelta. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.
Assuntos
Proteínas de Ligação a DNA/química , Modelos Químicos , Modelos Moleculares , Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos/química , Ribossomos/química , Tricosantina/química , Sítios de Ligação , Simulação por Computador , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Angiotensin II (Ang II), a major regulator of blood pressure, is also involved in the control of cellular proliferation and hypertrophy and might exhibit additional actions in vivo by modulating the signaling of other hormones. As hypertension and Insulin (Ins) resistance often coexist and are risk factors for cardiovascular diseases, Ang II and Insulin signaling cross-talk may have an important role in hypertension development. The effect of Ins on protein tyrosine phosphorylation was assayed in rat liver membrane preparations, a rich source of Ins receptors. Following stimulation, Ins (10(-7) M) induced tyr-phosphorylation of different proteins. Insulin consistently induced tyr-phosphorylation of a 160 kDa protein (pp160) with maximum effect between 1 and 3 min. The pp160 protein was identified by anti-IRS-4 but not by anti-IRS-1 antibody. Pre-stimulation with Ang II (10(-7) M) diminishes tyr-phosphorylation level of pp160/IRS-4 in a dose-dependent manner. Okadaic acid, the PP1A and PP2A Ser/Thr phosphatase inhibitor, increases pp160 phosphorylation induced by Ins and prevents the inhibitory effect of Ang II pre-stimulation. Genistein, a tyrosine kinase inhibitor, diminishes tyr-phosphorylation level of IRS-4. PI3K inhibitors Wortmanin and LY294002, both increase tyr-phosphorylation of IRS-4, either in the presence of Ins alone or combined with Ang II. These results suggest that Ins and Ang II modulate IRS-4 tyr-phosphorylation in a PI3K-dependent manner. In summary, we showed that Ins induces tyr-phosphorylation of IRS-4, an effect modulated by Ang II. Assays performed in the presence of different inhibitors points toward a PI3K involvement in this signaling pathway.
Assuntos
Angiotensina II/farmacologia , Membrana Celular/enzimologia , Fígado/metabolismo , Fosfoproteínas/metabolismo , Tirosina/metabolismo , Vasoconstritores/farmacologia , Androstadienos/farmacologia , Angiotensina II/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Genisteína/farmacologia , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Fígado/efeitos dos fármacos , Masculino , Ácido Okadáico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Vasoconstritores/metabolismo , WortmaninaRESUMO
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 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 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 alpha/beta 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.
Assuntos
Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Trypanosoma cruzi/genéticaRESUMO
We present analysis, by cryo-electron microscopy and single-particle reconstruction, of the structure of the 80S ribosome from Trypanosoma cruzi, the kinetoplastid protozoan pathogen that causes Chagas disease. The density map of the T. cruzi 80S ribosome shows the phylogenetically conserved eukaryotic rRNA core structure, together with distinctive structural features in both the small and large subunits. Remarkably, a previously undescribed helical structure appears in the small subunit in the vicinity of the mRNA exit channel. We propose that this rRNA structure likely participates in the recruitment of ribosome onto the 5' end of mRNA, in facilitating and modulating the initiation of translation that is unique to the trypanosomes.
Assuntos
Modelos Moleculares , RNA Ribossômico/genética , Ribossomos/química , Trypanosoma cruzi/genética , Animais , Pareamento de Bases , Microscopia Crioeletrônica , Processamento de Imagem Assistida por ComputadorRESUMO
Growth inhibitory activities and nutritional indices of catalpol (1), 8-O-acetylharpagide (2), and harpagide (3) were determinated in larvae and adults of Tribolium castaneum, respectively. Compound 1 produced a series of allelochemical effects probably related with the DNA synthesis. This iridoid possessed the highest inhibitory activity against DNA polymerase. Molecular orbital calculations suggest that a pi-pi charge transfer recognition model could explain the action of iridioids toward nucleic acid synthesis.