Assuntos

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Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

Assuntos

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Conformational changes are an essential feature for the function of some dynamic proteins. Understanding the mechanism of such motions may allow us to identify important properties, which may be directly related to the regulatory function of a protein. Also, this knowledge may be employed for a rational design of drugs that can shift the balance between active and inactive conformations, as well as affect the kinetics of the activation process. Here, the conformational changes in carboxyl-terminal Src kinase, the major catalytic repressor to the Src family of kinases, was investigated, and it was proposed as a functionally related hypothesis. A Cα Structure-Based Model (Cα-SBM) was applied to provide a description of the overall conformational landscape and further analysis complemented by detailed molecular dynamics simulations. As a first approach to Cα-SBM simulations, reversible transitions between active (closed) and inactive (open) forms were modeled as fluctuations between these two energetic basins. It was found that, in addition to the interdomain Carboxyl-terminal SRC Kinase (Csk) correlated motions, a conformational change in the αC helix is required for a complete conformational transition. The result reveals this as an important region of transition control and domain coordination. Restrictions in the αC helix region of the Csk protein were performed, and the analyses showed a direct correlation with the global conformational changes, with this location being propitious for future studies of ligands. Also, the Src Homology 3 (SH3) and SH3 plus Src Homology 2 (SH2) domains were excluded for a direct comparison with experimental results previously published. Simulations where the SH3 was deleted presented a reduction of the transitions during the simulations, while the SH3-SH2 deletion vanishes the Csk transitions, corroborating the experimental results mentioned and linking the conformational changes with the catalytic functionality of Csk. The study was complemented by the introduction of a known kinase inhibitor close to the Csk αC helix region where its consequences for the kinetic behavior and domain displacement of Csk were verified through detailed molecular dynamics. The findings describe the mechanisms involving the Csk αC helix for the transitions and also support the dynamic correlation between SH3 and SH2 domains against the Csk lobes and how local energetic restrictions or interactions in the Csk αC helix can play an important role for long-range motions. The results also allow speculation if the Csk activity is restricted to one specific conformation or a consequence of a state transition, this point being a target for future studies. However, the αC helix is revealed as a potential region for rational drug design.

Assuntos

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Plant survival depends on adaptive mechanisms that constantly rely on signal recognition and transduction. The predominant class of signal discriminators is receptor kinases, with a vast member composition in plants. The transduction of signals occurs in part by a simple repertoire of heterotrimeric G proteins, with a core composed of α-, ß-, and γ-subunits, together with a 7-transmembrane Regulator G Signaling (RGS) protein. With a small repertoire of G proteins in plants, phosphorylation by receptor kinases is critical in regulating the active state of the G-protein complex. This review describes the in vivo detected phosphosites in plant G proteins and conservation scores, and their in vitro corresponding kinases. Furthermore, recently described outcomes, including novel arrestin-like internalization of RGS and a non-canonical phosphorylation switching mechanism that drives G-protein plasticity, are discussed.

Assuntos

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Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

Assuntos

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PURPOSE: Expression microarrays are powerful technology that allows large-scale analysis of RNA profiles in a tissue; these platforms include underexploited detection scores outputs. We developed an algorithm using the detection score, to generate a detection profile of shared elements in retinoblastoma as well as to determine its transcriptomic size and structure. METHODS: We analyzed eight briefly cultured primary retinoblastomas with the Human transcriptome array 2.0 (HTA2.0). Transcripts and genes detection scores were determined using the Detection Above Background algorithm (DABG). We used unsupervised and supervised computational tools to analyze detected and undetected elements; WebGestalt was used to explore functions encoded by genes in relevant clusters and performed experimental validation. RESULTS: We found a core cluster with 7,513 genes detected and shared by all samples, 4,321 genes in a cluster that was commonly absent, and 7,681 genes variably detected across the samples accounting for tumor heterogeneity. Relevant pathways identified in the core cluster relate to cell cycle, RNA transport, and DNA replication. We performed a kinome analysis of the core cluster and found 4 potential therapeutic kinase targets. Through analysis of the variably detected genes, we discovered 123 differentially expressed transcripts between bilateral and unilateral cases. CONCLUSIONS: This novel analytical approach allowed determining the retinoblastoma transcriptomic size, a shared active transcriptomic core among the samples, potential therapeutic target kinases shared by all samples, transcripts related to inter tumor heterogeneity, and to determine transcriptomic profiles without the need of control tissues. This approach is useful to analyze other cancer or tissue types.

Assuntos

RESUMO

The objective of this study was to evaluate the productive impact of colibacillosis on laying hens and to investigate whether energetic metabolism and oxidative stress were involved in the pathogenesis of the disease. An experimental shed containing 270 laying hens of the Hy-Line lineage (32 weeks old) presented approximately 40% daily laying, and many birds presented with diarrhea and apathy followed by death. Necropsy revealed macroscopic lesions compatible with colibacillosis and infectious agent Escherichia coli was isolated from fecal samples of all birds in the infected group, as well as from tissue (ovary, liver and peritoneum). Sixteen chickens were selected for this study, divided into two groups: Control (animals without clinical alterations) and infected (with diarrhea and apathetic). E. coli isolates were subjected to the antimicrobial susceptibility testing according to the methodology approved by CLSI, 2018. This testing showed sensitivity to gentamicin, amoxicillin, norfloxacin and colistin. It was then determined that laying hens would be treated with norfloxacin (15 mg/kg) diluted in water offered at will to the birds for three days. Blood collections were performed via brachial vein after the diagnosis of E. coli (before starting treatment) and seven days after treatment. Three debilitated chickens died on the second day after initiating therapy. Before treatment, birds with clinical signs had higher levels of lipoperoxidation (LPO) and activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) than in the control group (asymptomatic animals). After treatment, LPO levels remained higher in birds that had clinical disease (infected group), whereas the activity of SOD and GPx enzymes did not differ between groups. Activity levels of creatine kinase (CK) and pyruvate kinase (PK) were higher in the group of chickens with clinical disease before treatment. Post-treatment, no differences were observed between groups in terms of CK; however, PK activity remained high in these animals. In the hens that died, there were lesions characteristic of avian colibacillosis, with ovary involvement, explaining the low laying activity of the birds at their peak of production. For 10 days after starting treatment, the percentage of laying increased to 90%. Therefore, we conclude that colibacillosis interferes with the phosphotransfer network by stimulating ATP production, in addition to causing oxidative stress of the birds during laying, that negatively affects health and productive efficiency.

Assuntos

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This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.

Assuntos

RESUMO

AIMS: The identification of novel targets to control inflammation in humans is probably the primary challenge that impairs the development of new anti-inflammatory drugs. Therefore, the modulation of intracellular signaling pathways in phagocytes may be an interesting means of achieving this goal. However, this change to signaling can compromise the host's susceptibility to invading pathogens. We investigated whether the antioxidant nitroxide Tempol regulates the activity of kinases associated with the production of oxidants in neutrophils, which affects the fungicidal capability of these cells. MAIN METHODS: The effects of Tempol on PMA- or fMLP-activated neutrophils were examined by oxygen consumption as an index of the oxidative burst, a release of extracellular and total Reactive Oxygen Species (ROS) by chemiluminescence, kinase activities through analysis of ATP consumption during enzyme activities and the dot blot immunoassay and, finally, by neutrophil capacity of killing Candida albicans. KEY FINDINGS: Tempol significantly inhibited the neutrophil oxidative burst in a concentration-dependent manner and decreased oxygen consumption (IC50 = 45 µM) and extracellular/total ROS formation with an increase on the lag period response. In addition, Tempol inhibited neutrophil kinase activities (i.e., a decrease in protein phosphorylation) elicited through different biochemical pathways and consequently impaired the fungicidal activity of these cells. SIGNIFICANCE: Although Tempol has potential anti-inflammatory activity that acts on different intracellular pathways (such as those involving kinases), researchers should be cautious, since this nitroxide down-regulated oxidants production and the fungicidal response of neutrophils.

Assuntos

RESUMO

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.

Assuntos