Search

The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.

The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D-DIGE is carried out in four different groups (NT × T; NT × Se-NT (where Se is selenium); Se-NT × Se-T, and T × Se-T). Although no differential proteins are achieved in the T × Se-T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI-MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway-Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.

Nature Communications 5: Article number: 5519 (2014); Published: 16 October 2014; Updated: 23 March 2015 An image in Supplementary Fig. 11a in this Article, representing the negative control, was inadvertently duplicated from the ‘myc-FAK’-transfected group. The correct version of the figure appearsbelow.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

Background Plant development is very plastic, being coupled to environmental cues. As sessile organisms, plants must be able to respond rapidly to environmental stresses such as changes in temperature and salinity, heavy metals and water deficit. Efficient stress response systems are prerequisites for plant survival and productivity [1]. Molecular chaperones (or Heat Shock Proteins – HSP) compose a ubiquitous class of proteins involved in cellular protein quality control (PQC) and homeostasis. They play a critical role in folding and degradation of polypeptides, and therefore, in maintenance and modulation of cellular pathways, which are dependent of function (correct folding) and availability (stability and degradation) of involved proteins, under normal and stress conditions [2]. Genetics and proteomics studies of wood formation have highlighted some chaperones up-regulated in xylem of Eucalyptus, Pinus and Populus species, stating that they may play an important role in cell wall formation and xylem development [3,4]. Different species of Eucalyptus are known for their superior performance in growth, wood quality and resistance to different types of stress [5]. Such characteristics are probably driven by distinct gene expression coordination in xylogenesis. Eucalyptus grandis is one of the most planted species in the world due to its rapid growth, wide adaptability and wood quality. Eucalyptus globulus wood has higher S/G ratio which provides high yields in cellulose extraction [6]. Lignin extraction consumes large quantities of chemicals and energy, and many efforts have been made to improve this process by modifying lignin content or composition in trees, in order to reduce lignin content or make it easier to extract. Results have been achieved by supplementation and genetic modification [7,8]. This study aims to identify chaperones possibly involved in wood formation and quality of wood for pulp and paper industries.

Background The flavonoids, naringenin-chalcone and narigenin, are intermediates in phenylpropanoid metabolism in plants, occupying the central position as primary intermediates in flavonoid biosynthesis and are synthesized by chalcone synthase (CHS) and chalcone isomerase (CHI) respectively.[1] It has been reported that supplementation of narigenin-chalcone and narigenin can inhibit the activity of 4CL in vitro, and suppress the growth and reduce lignin content in gramineous plants, while 4CL suppression affected plant phenotype and resulted on dwarfed trees on Pinus[2], and its down-regulation promoted enhanced growth phenotypes on transgenic aspens trees [3], also CHS expression controls flavonoid synthesis and reduced size phenotype on arabidopsis. [4] Eucalyptus is the main source of biomass for pulp and paper industries therefore it’s imperative to study the influence of flavonoid supplementation on Eucalyptus and what kind of overall impact it can have on plant development, especially wood formation and gene expression.

Background In Brazil, the forest industry accounts for 4,5% from the Gross Domestic Product and the country is the biggest Eucalyptus cellulose exporter. That’s really good news because Eucalyptus forests are a competitive and efficient alternative to convert carbon from the atmosphere in cellulose, an important source for paper and bioenergy production. The cellulose biosynthesis happens through the Cellulose Synthase Complex activity. This complex is composed by different Cellulose Synthase genes (CesA) that work together in a non redundant way [1]. It is also known that some of these isoforms act in the primary cell wall synthesis while others, in the secondary cell wall. In this last group there is the gene Eucalyptus Cellulose Synthase 3 (EgCesA3), the most expressed CesA gene during xylogenesis [2]. Besides, knockout experiments proved that the AtCesA7 (EgCesA3 ortholog in Arabidopsis thaliana) activity is essential for the xylem formation and for plant vertical growth [3]. In front of these evidences, the EgCesA3 gene had it expression pattern evaluated in leaf and xylem tissues among the three most economic important Eucalyptus species in Brazil: E. grandis, E. globulus andE. urophyla. As demonstrated in this work, the expression experiment provided enough information about the EgCesA3 function, that’s why this gene was chosen to be overexpressed in model plants (Arabidopsis thaliana and Nicotiana tabacum). It’s expected increased cellulose content in the transgenic plants xylem.

Background The Eucalyptus wood is one of the most important raw materials for pulp and paper industry. Brazil is currently the first producer of short-fiber pulp and sixth in total production of cellulose. To maintain the industrial competitiveness, investment in genomic research started in 2002 with the GENOLYPTUS project (Brazilian Network of Eucalyptus Genome Research). Recently, a new transcriptome library was generated using Next Generation RNA Sequencing by Illumina’s sequencing by synthesis technology. Different species of Eucalyptus are recognized for their superior characteristics in terms of growth, wood quality and resistance to different types of stress (1). Such features are probably driven by the coordinated expression of numerous genes involved in processes of structural and regulatory genes in xylogenesis. Therefore, the main purpose of this study is to identify genes and key metabolic compounds directly involved in wood quality, as well as transcription factors involved. An extensive data mining in the RNAseq database was conducted to identify sequences over expressed in xylem and those that were differentially expressed between species.

Hop is a tetratricopeptide repeat domain (TPR)-containing co-chaperone that is able to directly associate with both Hsp70 and Hsp90. Previous data showed that the TPR2A-domain is the primary site for dimerization and that the TPR2B-domain may also play a role in dimerization. We present Hop-D456G, a mutant within the TPR2B-domain, that is a mixture of monomeric and dimeric species.

Background Eucalyptus is one of the most important sources of industrial cellulose. Three species of this botanical group are intensively used in breeding programs: E. globulus, E. grandis and E. urophylla. E. globulus is adapted to subtropical/temperate areas and is considered a source of high-quality cellulose; E. grandis grows rapidly and is adapted to tropical/subtropical climates; and E. urophylla, though less productive, is considered a source of genes related to robustness. Wood, or secondary xylem, results from cambium vascular differentiation and is mostly composed of cellulose, lignin and hemicelluloses. In this study, the xylem transcriptomes of the three Eucalyptus species were investigated in order to provide insights on the particularities presented by each of these species. Results Data analysis showed that (1) most Eucalyptus genes are expressed in xylem; (2) most genes expressed in species-specific way constitutes genes with unknown functions and are interesting targets for future studies; (3) relevant differences were observed in the phenylpropanoid pathway: E. grandis xylem presents higher expression of genes involved in lignin formation whereas E. urophylla seems to deviates the pathway towards flavonoid formation; (4) stress-related genes are considerably more expressed in E. urophylla, suggesting that these genes may contribute to its robustness. Conclusions The comparison of these three transcriptomes indicates the molecular signatures underlying some of their distinct wood characteristics. This information may contribute to the understanding of xylogenesis, thus increasing the potential of genetic engineering approaches aiming at the improvement of Eucalyptus forest plantations productivity.

Background Eucalyptus forests are a competitive and efficient alternative to convert carbon from the atmosphere in cellulose, an important source for paper manufacture and bioenergy production. To obtain transgenic Eucalyptus with important traits improved it is necessary to make modifications in genes that affect the final phenotype. One interesting gene that follows this requisite was recently found: this is the AtSHN2 gene (Arabidopsis thaliana SHINE 2). AtSHN2 codifies to a Transcription Factor known as “Arabidopsis SHINE/WAX INDUCER”. Instead of inducing drought tolerance in transgenic rice (Oryza sativa), AtSHN2 overexpression causes: i) 34% increase in the cellulose content; ii) 45% reduction in lignin content and iii) increase in wood digestibly (elevated S:G ratio) with no compromise in plant strength and performance [1]. The discovery of AtSHN2 function in plant cell wall formation, led Ambavaram and collaborators [1] to perform other studies and ultimately to propose the following model: AtSHN2 regulates positively MYB transcription factors (TF) related to cellulose synthesis and it downregulates MYBTF’s related to lignin formation. At the same time, SHINE can repress NAC TFthat controls MYB expression[1]. As a consequence of the interesting phenotype achieved through AtSHN2 overexpression in rice, this work focused on the identification and analyses of AtSHN orthologues in Eucalyptus. Bioinformatics tools were used to search for AtSHN similar genes in Eucalyptus. Moreover, the expression profile of the corresponding genes in Eucalyptus was evaluated to prove their role as AtSHN. To carry it on, the expression experiments were done with flower, leaf and xylem. If the Eucalyptus putativeSHINE’s has the same function of the AtSHN’s,, gene expression in flower tissues will be the highest [2]. This is because it is known that AtSHN’s genes are preferentially expressed in abscission and dehiscence zones, a phenomenon that usually occurs in lots of flower tissues.

A large majority of the 1000–1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a KD of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.

Background Eucalyptus species are the most widely planted hardwood trees in the world representing more than 4.75 million ha in Brazil. Their high productivity, valuable wood properties and wide adaptability could allow sustainable and cost-efficient production of lignocellulosic bioenergy. The main limitation to this objective is wood recalcitrance to degradation which is linked to the structure and composition of lignified secondary cell walls. Lignin, for example, impairs the accessibility of cellulose during kraft pulping as well as during saccharification, a key step of bioethanol production. The application of nitrogen fertilizers is one strategy to increase growth rates and productivity since nitrogen is one of the most limiting nutrient for tree growth and carbon sequestration. However, the effects of nitrogen availability on wood properties and related gene expression are poorly understood. In poplar, it was recently reported that N fertilization increased aerial biomass, while in wood, fibre morphology and secondary cell wall structure and composition were modified. An increase in cellulose coupled with a decrease in lignin was observed and the mRNA profiles evaluated by microarray showed that nitrogen and tension wood have overlapping effects [1]. Moreover, a highly significant genetic correlation was observed between plant growth and lignin/cellulose composition. Quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators [2]. In order to get an insight on the regulation of nitrogen availability on wood formation in Eucalyptus, we have studied the effects of nitrogen fertilization on xylem transcriptome profiles using RNA-seq technology.