Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 265
Filtrar
1.
Biochem J ; 477(5): 1049-1059, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32108856

RESUMO

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.


Assuntos
Proteínas de Arabidopsis/metabolismo , Sequência Conservada/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Lisina/metabolismo , Organelas/metabolismo , Timina/análogos & derivados , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Polimerase Dirigida por DNA/genética , Lisina/genética , Organelas/genética , Timina/metabolismo
2.
Mol Phylogenet Evol ; 144: 106713, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31863901

RESUMO

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.


Assuntos
Sequência Conservada/fisiologia , Evolução Molecular , Genomas de Plastídeos/genética , Rhodiola/classificação , Rhodiola/genética , Sequência de Bases , Crassulaceae/classificação , Crassulaceae/genética , Ecossistema , Variação Genética/fisiologia , Genoma de Planta/fisiologia , Filogenia , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico/genética , Tibet
3.
Nat Commun ; 10(1): 1189, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867425

RESUMO

In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/metabolismo , Crista Neural/embriologia , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Linhagem Celular , Sequência Conservada/fisiologia , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/genética , Lampreias , Camundongos , Células-Tronco Embrionárias Murinas , Crista Neural/metabolismo , Alinhamento de Sequência , Vertebrados/embriologia , Peixe-Zebra
4.
Mol Pharmacol ; 94(4): 1232-1245, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30111649

RESUMO

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.


Assuntos
Regulação Alostérica/fisiologia , Sítio Alostérico/fisiologia , Receptores de Dopamina D1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Linhagem Celular , Sequência Conservada/efeitos dos fármacos , Sequência Conservada/fisiologia , Dopamina/metabolismo , Células HEK293 , Humanos , Isoquinolinas/farmacologia , Ratos
5.
BMC Plant Biol ; 18(1): 129, 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29929474

RESUMO

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.


Assuntos
Proteínas de Domínio MADS/genética , Magnoliopsida/crescimento & desenvolvimento , Proteínas de Plantas/genética , Tabaco/crescimento & desenvolvimento , Evolução Biológica , Sequência Conservada/genética , Sequência Conservada/fisiologia , Genes de Plantas/genética , Genes de Plantas/fisiologia , Proteínas de Domínio MADS/fisiologia , Magnoliopsida/genética , Petunia/genética , Petunia/fisiologia , Filogenia , Proteínas de Plantas/fisiologia , Tabaco/genética , Transcriptoma
6.
Sci Rep ; 8(1): 4975, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563520

RESUMO

There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.


Assuntos
Sequência Conservada/fisiologia , Fertilidade/fisiologia , Proteínas/fisiologia , Serina Proteases/fisiologia , Espermatogênese/fisiologia , Animais , Sistemas CRISPR-Cas/genética , Sequência Conservada/genética , Epididimo/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas/genética , Serina Proteases/genética , Contagem de Espermatozoides , Testículo/fisiologia
7.
BMC Plant Biol ; 17(1): 212, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29157210

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.


Assuntos
Camellia sinensis/genética , Sequência Conservada/genética , MicroRNAs/genética , Folhas de Planta/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , RNA Interferente Pequeno/genética , Camellia sinensis/crescimento & desenvolvimento , Sequência Conservada/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genoma de Planta/genética , Genoma de Planta/fisiologia , Estudo de Associação Genômica Ampla , MicroRNAs/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/genética , Brotos de Planta/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
8.
Biochem Pharmacol ; 144: 35-51, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28774731

RESUMO

Influenza A viruses (IAVs) induce acute respiratory disease and cause significant morbidity and mortality throughout the world. With the emergence of drug-resistant viral strains, new and effective anti-IAV drugs with different modes of action are urgently needed. In this study, by conjugating cholesterol to the N-terminus of the short peptide KKWK, a lipopeptide named S-KKWK was created. The anti-IAV test indicated that S-KKWK and its derivatives displayed potent antiviral activities against a broad variety of influenza A viral strains including oseltamivir-resistant strains and clinically relevant isolates with IC50 values ranging from 0.7 to 3.0µM. An extensive mechanistic study showed that these peptides functioned as viral "entry blockers" by inhibiting the conformational rearrangements of HA2 subunit, thereby interrupting the fusion of virus-host cell membranes. Significantly, a computer-aided docking simulation and protein sequence alignment identified conserved residues in the stem region of HA2 as the possible binding site of S-KKWK, which may be employed as a potential drug target for designing anti-IAVs with a broad-spectrum of activity. By targeting this region, a potent anti-IAV agent was subsequently created. In addition, the anti-IAV activity of S-KKWK was assessed by experiments with influenza A virus-infected mice, in which S-KKWK reduced the mortality of infected animals and extended survival time significantly. Overall, in addition to providing a strategy for designing broad-spectrum anti-IAV agents, these results indicate that S-KKWK and its derivatives are prospective candidates for potent antivirals.


Assuntos
Antivirais/metabolismo , Sequência Conservada/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Hemaglutininas/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/metabolismo , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antivirais/administração & dosagem , Galinhas , Sequência Conservada/fisiologia , Cães , Hemaglutininas/genética , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
10.
Parasit Vectors ; 10(1): 251, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28526096

RESUMO

BACKGROUND: Adhesin proteins are used by Plasmodium parasites to bind and invade target cells. Hence, characterising molecules that participate in reticulocyte interaction is key to understanding the molecular basis of Plasmodium vivax invasion. This study focused on predicting functionally restricted regions of the P. vivax GPI-anchored micronemal antigen (PvGAMA) and characterising their reticulocyte binding activity. RESULTS: The pvgama gene was initially found in P. vivax VCG-I strain schizonts. According to the genetic diversity analysis, PvGAMA displayed a size polymorphism very common for antigenic P. vivax proteins. Two regions along the antigen sequence were highly conserved among species, having a negative natural selection signal. Interestingly, these regions revealed a functional role regarding preferential target cell adhesion. CONCLUSIONS: To our knowledge, this study describes PvGAMA reticulocyte binding properties for the first time. Conserved functional regions were predicted according to natural selection analysis and their binding ability was confirmed. These findings support the notion that PvGAMA may have an important role in P. vivax merozoite adhesion to its target cells.


Assuntos
Sequência Conservada/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reticulócitos/parasitologia , Seleção Genética , Antígenos de Protozoários/genética , Antígenos de Protozoários/fisiologia , Adesão Celular , Variação Genética , Plasmodium vivax/genética , Polimorfismo Genético , Ligação Proteica , Análise de Sequência de DNA
11.
Artigo em Inglês | MEDLINE | ID: mdl-26812730

RESUMO

Knowing the ways proteins interact with each other are crucial to our understanding of the functional mechanisms of proteins. It is for this reason that different approaches have been developed in attempts to predict protein-protein interactions (PPIs) computationally. Among them, the sequence-based approaches are preferred to the others as they do not require any information about protein properties to perform their tasks. Instead, most sequence-based approaches make use of feature extraction methods to extract features directly from protein sequences so that for each protein sequence, we can construct a feature vector. The feature vectors of every pair of proteins are then concatenated to form two classes of interacting and non-interacting proteins. The prediction of whether or not two proteins interact with each other is then formulated as a classification problem. How accurate PPI predictions can be made therefore depends on how good the features are that can be extracted from the protein sequences to allow interacting or non-interacting to be best distinguished. To do so, instead of extracting such features from individual protein sequences independently of the other protein in the same pair, we propose to jointly consider features from both sequences in a protein pair during the feature extraction process through using a novel coevolutionary feature extraction approach called CoFex. Coevolutionary features extracted by CoFex refer to the covariations found at coevolving positions. Based on the presence and absence of these coevolutionary features in the sequences of two proteins, feature vectors can be composed for pairs of proteins rather than individual proteins. The experiment results show that CoFex is a promising feature extraction approach and can improve the performance of PPI prediction.


Assuntos
Sequência Conservada/fisiologia , Evolução Molecular , Modelos Genéticos , Mapeamento de Interação de Proteínas/métodos , Proteoma/química , Proteoma/metabolismo , Análise de Sequência de Proteína/métodos , Sítios de Ligação , Simulação por Computador , Modelos Químicos , Ligação Proteica
12.
Biochim Biophys Acta Gen Subj ; 1861(2): 97-105, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27825831

RESUMO

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Fusão de Membrana/fisiologia , Membranas/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Aminoácidos/metabolismo , Sequência Conservada/fisiologia , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica/fisiologia , Conformação Proteica
13.
PLoS Comput Biol ; 12(11): e1004999, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27870845

RESUMO

Conserved moieties are groups of atoms that remain intact in all reactions of a metabolic network. Identification of conserved moieties gives insight into the structure and function of metabolic networks and facilitates metabolic modelling. All moiety conservation relations can be represented as nonnegative integer vectors in the left null space of the stoichiometric matrix corresponding to a biochemical network. Algorithms exist to compute such vectors based only on reaction stoichiometry but their computational complexity has limited their application to relatively small metabolic networks. Moreover, the vectors returned by existing algorithms do not, in general, represent conservation of a specific moiety with a defined atomic structure. Here, we show that identification of conserved moieties requires data on reaction atom mappings in addition to stoichiometry. We present a novel method to identify conserved moieties in metabolic networks by graph theoretical analysis of their underlying atom transition networks. Our method returns the exact group of atoms belonging to each conserved moiety as well as the corresponding vector in the left null space of the stoichiometric matrix. It can be implemented as a pipeline of polynomial time algorithms. Our implementation completes in under five minutes on a metabolic network with more than 4,000 mass balanced reactions. The scalability of the method enables extension of existing applications for moiety conservation relations to genome-scale metabolic networks. We also give examples of new applications made possible by elucidating the atomic structure of conserved moieties.


Assuntos
Sequência Conservada/fisiologia , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Modelos Químicos , Proteoma/química , Proteoma/metabolismo , Simulação por Computador , Homologia de Sequência de Aminoácidos
14.
J Exp Bot ; 67(18): 5447-5460, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27574185

RESUMO

DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A-green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A-GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast-ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome.


Assuntos
Sementes/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Sequência Conservada/genética , Sequência Conservada/fisiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Plastídeos/genética , Plastídeos/fisiologia , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Ribossomos/genética , Ribossomos/fisiologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Tabaco/genética , Tabaco/crescimento & desenvolvimento
15.
J Bacteriol ; 198(16): 2263-74, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27297880

RESUMO

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.


Assuntos
Proteínas de Bactérias/metabolismo , Sequência Conservada/fisiologia , AMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , AMP Cíclico/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Filogenia , Conformação Proteica , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo II
16.
J Exp Bot ; 67(9): 2641-53, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26951373

RESUMO

In plants, miRNAs are endogenous small RNAs derived from single-stranded precursors with hairpin structures. The evolution of miRNAs and their targets represents one of the most dynamic circuits directing gene expression, which may play fundamental roles in shaping the development of distinct plant organs. Here we performed high-throughput small RNA sequencing in five organ types of Camellia azalea to capture the spatial profile of small non-coding RNA. In total we obtained >227 million high-quality reads and identified 175 miRNAs with mature and precursor sequences. We aligned the miRNAs to known miRNA databases and revealed some conserved as well as 'newly evolved' miRNA genes. Twelve miRNAs were identified to be specific in the genus Camellia, supporting the lineage-specific manner of expansion of 'young' miRNAs. Through differential expression analysis, we showed that many miRNAs were preferentially abundant in certain organ types. Moreover, hierarchical clustering analysis revealed distinctive expression patterns of tissue-specific miRNAs. Gene Ontology enrichment analysis of targets of stamen- and carpel-specific miRNA subclusters showed that miRNA-target regulatory circuits were involved in many important biological processes, enabling their proper specification and organogenesis, such as 'DNA integration' and 'fruit development'. Further, quantitative PCR of key miRNAs and their target genes revealed anti-correlated patterns, and uncovered the functions of key miRNA-target pairs in different floral organs. Taken together, this work yielded valuable information on miRNA-target regulation in the control of floral organ development and sheds light on the evolution of lineage-specific miRNAs in Camellia.


Assuntos
Camellia/crescimento & desenvolvimento , Sequência Conservada/genética , Flores/crescimento & desenvolvimento , MicroRNAs/genética , Camellia/genética , Sequência Conservada/fisiologia , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/fisiologia , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
17.
PLoS One ; 11(3): e0151934, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26986966

RESUMO

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such "Cys-less" receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the "Cys-loop" proline is absolutely conserved, suggesting the more fitting name "Pro loop" for that motif, and "Pro-loop receptors" for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.


Assuntos
Canais Iônicos de Abertura Ativada por Ligante/genética , Receptores de Neurotransmissores/genética , Animais , Archaea/classificação , Archaea/genética , Archaea/fisiologia , Sequência Conservada/genética , Sequência Conservada/fisiologia , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/fisiologia , Eucariotos/genética , Eucariotos/fisiologia , Evolução Molecular , Fungos/genética , Fungos/fisiologia , Invertebrados/genética , Invertebrados/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Filogenia , Plantas/genética , Receptores de Neurotransmissores/fisiologia , Alinhamento de Sequência
18.
PLoS Negl Trop Dis ; 9(9): e0004099, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26398185

RESUMO

BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel ß-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope. CONCLUSIONS/SIGNIFICANCE: Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.


Assuntos
Glicoproteínas/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Filamentos Intermediários/metabolismo , Laminina/metabolismo , Neuraminidase/metabolismo , Estrutura Terciária de Proteína/fisiologia , Trypanosoma cruzi/metabolismo , Motivos de Aminoácidos/fisiologia , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Cromatografia de Afinidade , Sequência Conservada/fisiologia , Glicoproteínas/química , Humanos , Queratinas/metabolismo , Laminina/química , Neuraminidase/química , Ligação Proteica , Vimentina/metabolismo
19.
Plant Cell ; 27(6): 1605-19, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26036253

RESUMO

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.


Assuntos
Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Zea mays/genética , Sequência Conservada/genética , Sequência Conservada/fisiologia , Reguladores de Crescimento de Planta/genética , Reguladores de Crescimento de Planta/fisiologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Espectrometria de Massas em Tandem
20.
Ann N Y Acad Sci ; 1362: 48-56, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26104486

RESUMO

Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire.


Assuntos
Formação de Anticorpos/fisiologia , Subpopulações de Linfócitos B/fisiologia , Regiões Determinantes de Complementaridade/fisiologia , Sequência Conservada/fisiologia , Evolução Molecular , Células Germinativas/fisiologia , Animais , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...