RESUMO
Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function.
Assuntos
Íntrons , RNA de Transferência de Tirosina/metabolismo , Trypanosoma brucei brucei/metabolismo , AnimaisRESUMO
Aminoacyl-tRNA synthetases aaRSs are responsible for the aminoacylation of tRNAs in the first step of protein synthesis. They comprise a group of enzymes that catalyze the formation of each possible aminoacyl-tRNA necessary for messenger RNA decoding in a cell. These enzymes have been divided into two classes according to structural features of their active sites and, although each class shares a common active site core, they present an assorted array of appended domains that makes them sufficiently diverse among the different living organisms. Here we will explore what is known about the diversity encountered among trypanosomatids' aaRSs that has helped us not only to understand better the biology of these parasites but can be used rationally for the design of drugs against these protozoa.
Assuntos
Aminoacil-tRNA Sintetases/biossíntese , Trypanosoma/enzimologia , Aminoacil-tRNA Sintetases/química , Animais , Modelos MolecularesRESUMO
Vector-borne diseases are globally prevalent and represent a major socioeconomic problem worldwide. Blood-sucking arthropods transmit most pathogenic agents that cause these human infections. The pathogens transmission to their vertebrate hosts depends on how efficiently they infect their vector, which is particularly impacted by the microbiota residing in the intestinal lumen, as well as its cells or internal organs such as ovaries. The balance between costs and benefits provided by these interactions ultimately determines the outcome of the relationship. Here, we will explore aspects concerning the nature of microbe-vector interactions, including the adaptive traits required for their establishment, the varied outcomes of symbiotic interactions, as well as the factors influencing the transition of these relationships across a continuum from parasitism to mutualism.
Assuntos
Vetores Artrópodes , Simbiose , Animais , Vetores Artrópodes/microbiologia , Vetores Artrópodes/parasitologia , Insetos Vetores/microbiologia , Insetos Vetores/fisiologia , Doenças Transmitidas por Vetores/transmissãoRESUMO
The multidrug-resistant (MDR) phenotype is multifactorial, and cell lines presenting multiple resistance mechanisms might be good models to understand the importance of the various pathways involved. The present work characterized a MDR chronic myeloid leukemia cell line, derived from K562 through a selective process using daunorubicin. This MDR cell line was shown to be resistant to vincristine, daunorubicin, and partially resistant to imatinib. It showed a slower duplication rate. Overexpression of ABCB1 and ABCC1 was observed at the protein and functional levels and the expression of CD95, a molecule related to cell death, was reduced in the MDR cell line. Conversely, no differences were observed related to the anti-apoptotic molecule Bcl-2 or p53 expression. The activation antigen CD69 was reduced in the MDR cell line and treatment with imatinib further decreased the expressed levels. Furthermore, secretion of IL-8 was diminished in the MDR cell line. When daunorubicin-selected cells were compared to another MDR cell line, Lucena 1, derived from the same parental line K562, and selected with vincristine, a different profile was observed in relation to most aspects studied. When both cell lines were silenced for ABCB1, differences in CD69 and CD95 were maintained, despite resistance reversal. These results reinforce the idea that cell lines selected in vitro may display multiple resistance strategies that may vary with the selective agent used as well as during different steps of the selection process.
Assuntos
Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Inativação Gênica/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Lectinas Tipo C/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fenótipo , Receptor fas/metabolismoRESUMO
Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H(2)O(2) formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Estágios do Ciclo de Vida/fisiologia , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Complexo II de Transporte de Elétrons/imunologia , Complexo III da Cadeia de Transporte de Elétrons/imunologia , Humanos , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologiaRESUMO
Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.
RESUMO
Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.
Assuntos
Proteínas Reguladoras de Apoptose/química , Regulação da Expressão Gênica , RNA Bacteriano/genética , RNA de Transferência/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Escherichia coli/genética , Humanos , Transição de Fase , Conformação Proteica , RNA Bacteriano/química , RNA de Transferência/químicaRESUMO
Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Desulfitobacterium/enzimologia , Lisina/análogos & derivados , RNA de Transferência/metabolismo , Aminoacilação , Sequência de Bases , Iodo/química , Lisina/metabolismo , Dados de Sequência Molecular , Pegadas de Proteínas , RNA de Transferência/química , RibonucleasesRESUMO
Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.
Assuntos
Lisina/análogos & derivados , Methanosarcina barkeri/metabolismo , RNA Arqueal/metabolismo , RNA de Transferência de Lisina/metabolismo , Aminoacilação de RNA de Transferência , Alanina/química , Alanina/metabolismo , Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lisina/química , Lisina/metabolismo , Methanosarcina barkeri/enzimologia , Methanosarcina barkeri/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , RNA Arqueal/química , RNA de Transferência de Lisina/química , Serina/química , Serina/metabolismo , Serina-tRNA Ligase/química , Serina-tRNA Ligase/metabolismoRESUMO
Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNA(Pyl), which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA(Pyl). Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA(Pyl) with higher affinity (K(D)=0.1-1.0 microM) than D. hafniense PylRS (K(D)=5.3-6.9 microM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA(Pyl) species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.
Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/fisiologia , Lisina/análogos & derivados , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Sequência de Bases , Desulfitobacterium/enzimologia , Desulfitobacterium/genética , Ativação Enzimática , Variação Genética , Lisina/metabolismo , Methanosarcina/enzimologia , Methanosarcina/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context. We show that PYLIS has no effect on the level of neither Pyl nor Cyc incorporation. Exogenously supplying our reporter system with d-ornithine significantly increases suppression efficiency, indicating that d-ornithine is a direct precursor to Pyl.
Assuntos
Escherichia coli/genética , Código Genético , Lisina/análogos & derivados , Proteínas Arqueais/genética , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/metabolismo , Genes Arqueais , Genes Bacterianos , Lisina/genética , Lisina/metabolismo , Methanosarcina barkeri/genética , Metiltransferases/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ornitina/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , Homologia de Sequência do Ácido NucleicoRESUMO
In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA(Pyl). We report here the in vitro aminoacylation of tRNA(Pyl) by PylRS with two Pyl analogues: N-epsilon-d-prolyl-l-lysine (d-prolyl-lysine) and N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc). Escherichia coli, transformed with the tRNA(Pyl) and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA(Pyl) and d-prolyl-lysine-tRNA(Pyl) as substrates during protein synthesis. Furthermore, the formation of active beta-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Lisina/análogos & derivados , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Lisina/química , Lisina/metabolismo , RNA Mensageiro , Especificidade por Substrato , Fatores de Tempo , Triptofano Sintase/metabolismo , beta-Galactosidase/metabolismoRESUMO
The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.
Assuntos
Aspartato-tRNA Ligase/metabolismo , Halobacterium salinarum/enzimologia , Aspartato-tRNA Ligase/genética , Proteínas de Bactérias , Halobacterium salinarum/genética , Dados de Sequência Molecular , Mutação , Transferases de Grupos Nitrogenados , RNA de Transferência de Asparagina , Especificidade por Substrato/genéticaRESUMO
The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrogenase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus.
Assuntos
Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Rhodnius/enzimologia , Rhodnius/genética , Selênio/química , Animais , Feminino , Inativação Metabólica/genética , Muda , Filogenia , Interferência de RNA , Coelhos , Rhodnius/crescimento & desenvolvimento , Selenocisteína/químicaRESUMO
All organisms encode transfer RNAs (tRNAs) that are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends; some tRNAs also contain introns, which are removed by splicing. Despite commonality in what the ultimate goal is (i.e., producing a mature tRNA), mechanistically, tRNA splicing differs between Bacteria and Archaea or Eukarya. The number and position of tRNA introns varies between organisms and even between different tRNAs within the same organism, suggesting a degree of plasticity in both the evolution and persistence of modern tRNA splicing systems. Here we will review recent findings that not only highlight nuances in splicing pathways but also provide potential reasons for the maintenance of introns in tRNA. Recently, connections between defects in the components of the tRNA splicing machinery and medically relevant phenotypes in humans have been reported. These differences will be discussed in terms of the importance of splicing for tRNA function and in a broader context on how tRNA splicing defects can often have unpredictable consequences.
Assuntos
Modelos Genéticos , Splicing de RNA , RNA de Transferência/química , Animais , Archaea/genética , Bactérias/genética , Endonucleases/fisiologia , Íntrons/fisiologia , Fosfotransferases/fisiologia , RNA de Transferência/metabolismo , RNA de Transferência/fisiologia , Vertebrados/genéticaRESUMO
Aminoacyl-tRNAs (aa-tRNAs) are essential substrates for ribosomal translation, and are generally synthesized by aminoacyl-tRNA synthetases (aaRSs). It was expected earlier that every organism would contain a complete set of twenty aaRSs, one for each canonical amino acid. However, analysis of the many known genome sequences and biochemical studies revealed that most organisms lack asparaginyl- and glutaminyl-tRNA synthetases, and thus are unable to attach asparagine and glutamine directly onto their corresponding tRNA. Instead, a pretranslational amino acid modification is required to convert Asp-tRNA(Asn) and Glu-tRNA(Gln) to the correctly charged Asn-tRNA(Asn) and Gln-tRNA(Gln), respectively. This transamidation pathway of amide aa-tRNA synthesis is common in most bacteria and archaea. Unexpected results from biochemical, genetic and genomic studies showed that a large variety of different bacteria rely on tRNA-dependent transamidation for the formation of the amino acid asparagine. Pretranslational modifications are not restricted to asparagine and glutamine but are also found in the biosynthesis of some other aa-tRNAs, such as the initiator tRNA fmet-tRNA(Met)(i) and Sec-tRNA(Sec) specifying selenocysteine, the 21(st) cotranslationally inserted amino acid. tRNA-dependent amino acid modification is also involved in the generation of aminolevulinic acid, the first precursor for porphyrin biosynthesis in many organisms.
Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Modificação Traducional de Proteínas , Aminoacil-RNA de Transferência , Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Archaea/metabolismo , Asparagina/química , Bacillus subtilis/metabolismo , Clorofila/química , Escherichia coli/metabolismo , Glutamina/química , Heme/química , Modelos Biológicos , Filogenia , Porfirinas/química , Especificidade da EspécieRESUMO
The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
Assuntos
Proteínas de Insetos/genética , Rhodnius/genética , Transcriptoma , Animais , Feminino , Trato Gastrointestinal , Proteínas de Insetos/biossíntese , América Latina , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA(Pyl) by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA(Pyl) in vivo by using the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. Forty-two Methanosarcina barkeri tRNA(Pyl) variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA(Pyl) mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA(Pyl) aminoacylation. tRNA(Pyl) identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA(Pyl) identity elements into the mitochondrial bovine tRNA(Ser) scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA(Pyl) variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA(Pyl) variants may decode numerous codons and that tRNA(Pyl):PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.
Assuntos
Lisina/análogos & derivados , Methanosarcina barkeri/genética , Biossíntese de Proteínas/genética , RNA de Transferência Aminoácido-Específico/genética , Aminoacilação de RNA de Transferência/genética , Aminoacil-tRNA Sintetases/metabolismo , Pareamento de Bases , Sequência de Bases , Códon/genética , Escherichia coli , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Mutação/genética , RNA de Transferência Aminoácido-Específico/metabolismo , Análise de Sequência de DNA , Supressão Genética/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNASer recognition, serylation of variant transcripts of M. barkeri tRNASer was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNASer recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNASer recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNASer recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNASer identity determinants reflects their evolutionary pathways, hence attesting to their diversity.
Assuntos
Methanosarcina barkeri/metabolismo , RNA de Transferência de Serina/metabolismo , Serina-tRNA Ligase/química , Serina-tRNA Ligase/genética , Sequência de Bases , Clonagem Molecular , Evolução Molecular , Cinética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA de Transferência/química , Serina/químicaRESUMO
Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.