Identification of Functional Subclasses in the DJ-1 Superfamily Proteins

Genomics has posed the challenge of determination of protein function from sequence and/or 3-D structure. Functional assignment from sequence relationships can be misleading, and structural similarity does not necessarily imply functional similarity. Proteins in the DJ-1 family, many of which are of unknown function, are examples of proteins with both sequence and fold similarity that span multiple functional classes. THEMATICS (theoretical microscopic titration curves), an electrostatics-based computational approach to functional site prediction, is used to sort proteins in the DJ-1 family into different functional classes. Active site residues are predicted for the eight distinct DJ-1 proteins with available 3-D structures. Placement of the predicted residues onto a structural alignment for six of these proteins reveals three distinct types of active sites. Each type overlaps only partially with the others, with only one residue in common across all six sets of predicted residues. Human DJ-1 and YajL from Escherichia coli have very similar predicted active sites and belong to the same probable functional group. Protease I, a known cysteine protease from Pyrococcus horikoshii, and PfpI/YhbO from E. coli, a hypothetical protein of unknown function, belong to a separate class. THEMATICS predicts a set of residues that is typical of a cysteine protease for Protease I; the prediction for PfpI/YhbO bears some similarity. YDR533Cp from Saccharomyces cerevisiae, of unknown function, and the known chaperone Hsp31 from E. coli constitute a third group with nearly identical predicted active sites. While the first four proteins have predicted active sites at dimer interfaces, YDR533Cp and Hsp31 both have predicted sites contained within each subunit. Although YDR533Cp and Hsp31 form different dimers with different orientations between the subunits, the predicted active sites are superimposable within the monomer structures. Thus, the three predicted functional classes form four different types of quaternary structures. The computational prediction of the functional sites for protein structures of unknown function provides valuable clues for functional classification

Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene

Background : The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF)which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. Results : A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters. Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. Conclusions : The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner

One origin for metallo-β-lactamase activity, or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees

This work was supported by BBSRC (grant BB/F016778/1)Bacteria use metallo-β-lactamase enzymes to hydrolyse lactam rings found in many antibiotics, rendering them ineffective. Metallo-β-lactamase activity is thought to be polyphyletic, having arisen on more than one occasion within a single functionally diverse homologous superfamily. Since discovery of multiple origins of enzymatic activity conferring antibiotic resistance has broad implications for the continued clinical use of antibiotics, we test the hypothesis of polyphyly further; if lactamase function has arisen twice independently, the most recent common ancestor (MRCA) is not expected to possess lactam-hydrolysing activity. Two major problems present themselves. Firstly, even with a perfectly known phylogeny, ancestral sequence reconstruction is error prone. Secondly, the phylogeny is not known, and in fact reconstructing a single, unambiguous phylogeny for the superfamily has proven impossible. To obtain a more statistical view of the strength of evidence for or against MRCA lactamase function, we reconstructed a sample of 98 MRCAs of the metallo-β-lactamases, each based on a different tree in a bootstrap sample of reconstructed phylogenies. InterPro sequence signatures and homology modelling were then used to assess our sample of MRCAs for lactamase functionality. Only 5 % of these models conform to our criteria for metallo-β-lactamase functionality, suggesting that the ancestor was unlikely to have been a metallo-β-lactamase. On the other hand, given that ancestral proteins may have had metallo-β-lactamase functionality with variation in sequence and structural properties compared with extant enzymes, our criteria are conservative, estimating a lower bound of evidence for metallo-β-lactamase functionality but not an upper bound.Publisher PDFPeer reviewe

A study of molecular responses to abiotic and biotic stresses in Arachis

Em seu ambiente natural, as plantas são expostas a uma série de estresses abióticos e bióticos. Entre eles, a seca é um grande obstáculo à agricultura e, em associação com patógenos e pragas, pode causar prejuízos de até US$38 bilhões de 2005 a 2015, e comprometer a segurança alimentar. Com as mudanças climáticas e aquecimento global, esse cenário tende a agravar-se, aumentando a ocorrência de secas, a amplitude da gama de hospedeiros e virulência de agentes patogênicos. O estudo da resposta molecular à estresses individuais em diferentes plantas assim como a resposta a estresses combinados, constitui um grande avanço, para a compreensão de possíveis trade-offs entre tolerância e suscetibilidade à estresses abióticos e bióticos, quanto o desenvolvimento de cultivares mais resistentes. Visando um melhor entendimento da resposta moleculares de plantas, o presente estudo empregou diferentes ferramentas, como genômica comparativa e transcritomas. O uso dessas ferramentas visou a identificação de genes comumente regulados por diferentes estresses. No capítulo I, o transcritoma de quatro diferentes espécies de plantas (Arachis stenosperma, Coffea arabica, Glycine max e Oryza glaberrima) inoculadas com nematoides das galhas (Meloidogyne spp.) foram analisados e combinados com dados de genômica comparativa gerados a partir do proteoma de 22 espécies de plantas. No total, 17 famílias de proteínas ortólogas são comumente reguladas pela inoculação de nematoides das galhas, representando ao todo 364 proteínas. A anotação funcional desses ortogrupos indicou que a maioria dessas proteínas está associada à parede celular, receptores quinases e a estresse, e tendo uma única família de fatores de transcrição representada, ERF a qual desempenha um papel fundamental na resposta de defesa da planta. No Capítulo II, a análise do transcritoma de Arachis stenosperma submetido à combinação de dois estresses, seca e inoculação por nematoides das galhas, foi realizada. Diversos genes de resistência a estresses combinados foram identificados sendo que uma resposta transcricional distinta foi observada quando da imposição de estresses combinados, quando comparada a estresses individuais. Apenas 14 genes foram encontrados em comum entre a seca, inoculação por nematoides e estresses combinados e 209 genes foram exclusivamente regulados no tratamento combinado. Este resultado demonstra que uma reprogramação molecular única é empregada pela planta sob múltiplos estresses, o que não pode ser previsto pela análise de cada estresse aplicado individualmente. A análise de genômica comparativa e a análise de estresse combinado realizadas no presente estudo permitiram a identificação de famílias de genes essenciais à resistência / tolerância de plantas. A família das expansinas, a qual foi modulada durante a seca, infecção por RKN e UV, revelando uma importante família de genes em resposta a múltiplos estresses e em espécies distintas; e um gene de desidrina, cuja superexpressão em Arabidopsis revelou um importante trade-off entre estresses abióticos e bióticos.In their natural environment plants are exposed to a range of abiotic and biotic stresses. Among them, drought is a major constraint to agriculture and, in association with pathogen and pests, can cause losses of up to$38 billion from 2005 to 2015 and compromise food security. With the advent of climate change and global warming, this scenario tends to become worse, with an increase in drought occurrence and pathogen`s host range and virulence. Breeding programs are doing great efforts to find solutions to improve multiple stress resistance in plants, however, the trade-offs between abiotic stress tolerance and biotic stress susceptibility have hardly been investigated. Here, a comparative genomics study was conducted, which has the potential to identify putative evolutionarily conserved genes involved in universal defense mechanisms, to identify genes regulated in common by Meloidogyne spp infection in four plant species (Arachis stenosperma, Coffea arabica, Glycine max, and Oryza glaberrima). In total, 17 orthologs protein families which respond to the inoculation of RKN in the four species, comprising a total of 364 proteins. The functional annotation of these orthogroups indicated that the majority of these genes are associated with the cell wall, receptor kinases and stress related, with the transcription factor ERF playing a pivotal role in these defense responses. A number of multiple-stress resistant genes was identified by analyzing the transcriptome of a resistant wild Arachis species, A. stenosperma, under the combination of RKN and drought imposition. An overall distinct transcriptional response was observed for the combinatory stress imposition when compared to each individual stress, with only 14 genes found in common among drought, nematode infection and combined stresses and 209 exclusively regulated genes within the combined treatment. This result demonstrates that a unique molecular reprogramming is employed by the plant under multiple stresses, which cannot be predicted by the analysis of each individual stress alone. The comparative genomics and the cross-stress analysis conducted in this study enabled the identification of genes families which are essential to plant resistance/tolerance. The gene family of the expansins which was modulated during drought, RKN infection and UV, disclosing an important gene family in response to multiple stresses and across species; and a dehydrin gene, 13 which overexpression in Arabidopsis revealed an important trade-off between abiotic and biotic stresses

Diversity and evolution of the small multidrug resistance protein family

<p>Abstract</p> <p>Background</p> <p>Members of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins.</p> <p>Results</p> <p>A thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of <it>groEL </it>mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain alignments to Bacterial/Archaeal transporters (BAT), DMT, and MFS sequences supports SMR participation in multidrug transport evolution by identifying common TM domains.</p> <p>Conclusion</p> <p>Based on this study, PSMR sequences originated recently within both SUG and SMP clades through gene duplication events and it appears that SMR members may be evolving towards specific metabolite transport.</p

A Plant DJ-1 Homolog Is Essential for Arabidopsis thaliana Chloroplast Development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

<p>Abstract</p> <p>Background</p> <p>The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes <it>C. elegans nhr-67, Drosophila tailless </it>and <it>dissatisfaction</it>, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes <it>C. elegans fax-1 </it>and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members.</p> <p>Results</p> <p>We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind <it>in vivo</it>.</p> <p>Conclusion</p> <p>These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1 and NR2E3 subclasses. For the NR2E1 protein NHR-67, Asp-19 permits binding to AAGTCA half-sites, while Asn-19 permits binding to AGGTCA half-sites. The apparent conservation of DNA-binding properties between vertebrate and nematode NR2E receptors allows for the possibility of evolutionarily-conserved regulatory patterns.</p