Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that aB-crystallin interacts with and confers protection to FAK against calpainmediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the b4-b8 groove of aB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. aB-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 aB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of aB-crystallin by RNA interference. These studies define a role for aB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.
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.
Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
BackgroundEucalyptus 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.ResultsData 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.ConclusionsThe 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.
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 K(D) 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.
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