Stress-responsive Mitogen-activated Protein Kinase Research Articles

MAPK Phosphatase-5 is required for TGF-β signaling through a JNK-dependent pathway.

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute members of the dual-specificity family of protein phosphatases that dephosphorylate the MAPKs. MKP-5 dephosphorylates the stress-responsive MAPKs, p38 MAPK and JNK, and has been shown to promote tissue fibrosis. Here, we provide insight into how MKP-5 regulates the transforming growth factor-β (TGF-β) pathway, a well-established driver of fibrosis. We show that MKP-5-deficient fibroblasts in response to TGF-β are impaired in SMAD2 phosphorylation at canonical and non-canonical sites, nuclear translocation, and transcriptional activation of fibrogenic genes. Consistent with this, pharmacological inhibition of MKP-5 is sufficient to block TGF-β signaling, and that this regulation occurs through a JNK-dependent pathway. By utilizing RNA sequencing and transcriptomic analysis, we identify TGF-β signaling activators regulated by MKP-5 in a JNK-dependent manner, providing mechanistic insight into how MKP-5 promotes TGF-β signaling. This study elucidates a novel mechanism whereby MKP-5-mediated JNK inactivation is required for TGF-β signaling and provides insight into the role of MKP-5 in fibrosis.

Genome-wide identification of the MAPK gene family in turbot and its involvement in abiotic and biotic stress responses

The mitogen-activated protein kinase (MAPK) gene family performs crucial roles in cell division, migration, development, apoptosis, inflammatory response, and abiotic and biotic stress responses. However, very little information is available about the MAPKs in turbot (Scophthalmus maximus). In this study, 15 turbot MAPKs (SmMAPKs) were identified throughout the whole genome, and their basic chemical and physical properties and subcellular localization were illustrated. All SmMAPKs contained the serine/threonine protein kinases, catalytic domain (S_TKc, SMART00220). Phylogenetic analysis revealed that SmMAPKs were classified into three subfamilies, namely, c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. Conserved motif and gene structure analysis revealed high levels of conservation within and between phylogenetic subfamilies. Expression patterns of MAPKs in distinct tissues and under diverse abiotic and biotic stresses were examined using the published available RNA-seq data sets. As a result, SmMAPKs showed obviously tissue-specific expression. Furthermore, 7 and 10 candidate stress-responsive MAPK genes were detected under abiotic and biotic stresses, respectively, among which five common MAPK genes, namely, SmMAPK4 (ERK4), SmMAPK6 (ERK3), SmMAPK11 (p38β), SmMAPK12b (p38γ), and SmMAPK15 (ERK7/8) showed extremely significant responses to both abiotic and biotic stresses, demonstrating their potential functions in comprehensive antistress. These results demonstrate that MAPKs might play vital roles in response to various abiotic and biotic stresses in turbot, which would contribute to making scientific preventive measures to environmental changes in the process of farming and promoting the development of selective breeding for comprehensive stress resistance in turbot.

Defining the structure-activity relationship for a novel class of allosteric MKP5 inhibitors

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease.

Targeting Stress Sensor Kinases in Hepatocellular Carcinoma-Infiltrating Human NK Cells as a Novel Immunotherapeutic Strategy for Liver Cancer.

Natural killer (NK) cells may become functionally exhausted entering hepatocellular carcinoma (HCC), and this has been associated with tumor progression and poor clinical outcome. Hypoxia, low nutrients, immunosuppressive cells, and soluble mediators characterize the intratumor microenvironment responsible for the metabolic deregulation of infiltrating immune cells such as NK cells. HCC-infiltrating NK cells from patients undergoing liver resection for HCC were sorted, and genome-wide transcriptome profiling was performed. We have identified a marked general upregulation of gene expression profile along with metabolic impairment of glycolysis, OXPHOS, and autophagy as well as functional defects of NK cells. Targeting p38 kinase, a stress-responsive mitogen-activated protein kinase, we could positively modify the metabolic profile of NK cells with functional restoration in terms of TNF-α production and cytotoxicity. We found a metabolic and functional derangement of HCC-infiltrating NK cells that is part of the immune defects associated with tumor progression and recurrence. NK cell exhaustion due to the hostile tumor microenvironment may be restored with p38 inhibitors with a selective mechanism that is specific for tumor-infiltrating—not affecting liver-infiltrating—NK cells. These results may represent the basis for the development of a new immunotherapeutic strategy to integrate and improve the available treatments for HCC.

Trans-Fatty Acids as an Enhancer of Inflammation and Cell Death: Molecular Basis for Their Pathological Actions.

trans-Fatty acids (TFAs) are food-derived fatty acids that possess one or more trans double bonds between carbon atoms. Compelling epidemiological and clinical evidence has demonstrated the association of TFA consumption with various diseases, such as cardiovascular diseases, and neurodegenerative diseases. However, the underlying etiology is poorly understood since the mechanisms of action of TFAs remain to be clarified. Previous studies have shown that single treatment with TFAs induce inflammation and cell death, but to a much lesser extent than saturated fatty acids (SFAs) that are well established as a risk factor for diseases linked with inflammation and cell death, which cannot explain the particularly higher association of TFAs with atherosclerosis than SFAs. In our series of studies, we have established the role of TFAs as an enhancer of inflammation and cell death. We found that pretreatment with TFAs strongly promoted apoptosis induced by either extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from damaged cells, or DNA damaging-agents, including doxorubicin and cisplatin, thorough enhancing activation of the stress-responsive mitogen-activated protein (MAP) kinase p38/c-jun N-terminal kinase (JNK) pathways; pretreatment with SFAs or cis isomers of TFAs had only minor or no effect, suggesting the uniqueness of the pro-apoptotic role of TFAs among fatty acids. Our findings will provide an insight into understanding of the pathogenesis mechanisms, and open up a new avenue for developing prevention strategies and therapies for TFA-related diseases.

Exploring the Biological and Molecular Characteristics of Resistance to Fludioxonil in Sclerotinia sclerotiorum From Soybean in China.

Sclerotinia sclerotiorum is one of the most damaging and economically important necrotrophic plant pathogens, infecting more than 400 plant species globally. Although the phenylpyrrole fungicide fludioxonil has high activity against S. sclerotiorum, reports indicate that there is also substantial potential for the development of fungicide resistance. However, the current study investigating five fludioxonil-resistant laboratory mutants found a significant fitness cost associated with fludioxonil resistance resulting in significantly (P < 0.05) reduced mycelial growth and sclerotia formation on potato dextrose agar as well as significantly (P < 0.05) lower pathogenicity on detached tomato leaves, with one mutant, LK-1R, completely losing the capacity to cause infection. In addition, all of the fludioxonil-resistant mutants had significantly (P < 0.05) increased sensitivity to osmotic stress (0.5 M of potassium chloride and 1.0 M of glucose), which is consistent with the proposed fludioxonil target sites within the high osmolarity glycerol stress response mitogen-activated protein kinase (HOG1-MAPK) signaling transduction pathway. Sequence analysis of six genes from this two-component pathway, including SsHk, SsYpd, SsSk1, SsSk2, SsPbs, and SsHog, revealed several mutations that may be associated with fludioxonil resistance. For example, six separate point mutations were found in SsHk that led to changes in the predicted amino acid sequence, including A136G, F249V, G353A, E560K, M610K, and K727R. Similarly, SsPbs had three mutations (D34G, S46L, and L337E), SsSk1 and SsYpd had two (S53G and A795V for SsSk1, and E67G and Y141H for SsYpd), and SsHog and SsSk2 had one each (V220A and S763P, respectively). To our knowledge, these constitute the first reports of amino acid changes in proteins of the HOG1-MAPK pathway being associated with fludioxonil resistance in S. sclerotiorum. This study also showed a positive cross-resistance between fludioxonil and dimethachlone and procymidone, but none with tebuconazole or carbendazim, indicating that the inclusion of tebuconazole within an integrated pest management program could reduce the risk of fludioxonil resistance developing in field populations of S. sclerotiorum and ensure the sustainable production of soybeans in China into the future.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10–20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1–20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress–MAPK signaling–inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

Swertiamarin from Enicostemma littorale, counteracts PD associated neurotoxicity via enhancement α-synuclein suppressive genes and SKN-1/NRF-2 activation through MAPK pathway

The elusive targets and the multifactorial etiology of Parkinson’s disease (PD) have hampered the discovery of a potent drug for PD. Furthermore, the presently available medications provide only symptomatic relief and have failed to mitigate the pathogenesis associated with PD. Therefore, the current study was aimed to evaluate the prospective of swertiamarin (SW), a secoiridoid glycoside isolated from a traditional medicinal plant, Enicostemma littorale Blume to ameliorate the characteristic features of PD in Caenorhabditis elegans. SW (25 μM) administration decreased the α-synuclein (α-syn) deposition, inhibited apoptosis and increased dopamine level mediated through upregulating the expression of genes linked to ceramide synthesis, mitochondrial morphology and function regulation, fatty acid desaturase genes along with stress responsive MAPK (mitogen-activated protein kinase) pathway genes. The neuroprotective effect of SW was evident from the robust reduction of 6-hydroxydopamine (6-OHDA) induced dopaminergic neurodegeneration independent of dopamine transporter (dat-1). SW mediated translational regulation of MAPK pathway genes was observed through increase expression of SKN-1 and GST-4. Further, in-silico molecular docking analysis of SW with C. elegans MEK-1 showed a promising binding affinity affirming the in-vivo results. Overall, these novel finding supports that SW is a possible lead for drug development against the multi- factorial PD pathologies.

Elaidic Acid Potentiates Extracellular ATP-Induced Apoptosis via the P2X7-ROS-ASK1-p38 Axis in Microglial Cell Lines.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X7), or Ca2+/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

Communication between Cyclin-dependent kinase Cdc2 and the Wis1-Spc1 MAPK pathway determines mitotic timing in Schizosaccharomyces pombe.

ABSTRACTCheckpoint activation and gene expression modulation represent key determinants of cellular survival in adverse conditions. The former is regulated by cyclin-dependent kinases (CDKs) while the latter can be controlled by mitogen-activated protein kinases (MAPKs). Association between cell-cycle progression and MAPK-dependent gene expression exists in cells growing in optimal environments. While MAPK-mediated regulation of the cell cycle is well characterised, the reciprocal influence of mitotic CDK on stress response is not well studied. We present evidence that CDK activity can regulate the extent of MAPK activation in Schizosaccharomyces pombe cells. We show that increasing or decreasing mitotic CDK (Cdc2) activity in S. pombe cells can affect the activation of stress responsive MAPK (Spc1) even in the absence of stress stimuli. Our results indicate that the strong correlation between Cdc2 activity and Spc1 MAPK-activity in S. pombe is important in regulating mitotic timing.This article has an associated First Person interview with the first author of the paper.

TORC1, Tel1/Mec1, and Mpk1 regulate autophagy induction after DNA damage in budding yeast

Target of rapamycin complex 1 (TORC1) protein kinase responds to various stresses including genotoxic stress. However, its molecular mechanism is poorly understood. Here, we show that DNA damage induces nonselective and selective autophagy in budding yeast. DNA damage caused the attenuation of TORC1 activity, dephosphorylation of Atg13, and autophagy induction. The TORC1-upstream Rag GTPase Gtr1 was not required for TORC1 inactivation and autophagy induction after DNA damage. Furthermore, DNA damage responsive protein kinases Mec1/ATM and Tel1/ATR, and stress-responsive mitogen-activated protein kinase Mpk1/Slt2 were required for the full induction of autophagy. Autophagic proteolysis was required for DNA damage tolerance in TORC1 inactive conditions. This study revealed that multiple protein kinases regulate DNA damage-induced autophagy.

Skeletal Muscle–Specific Deletion of MKP-1 Reveals a p38 MAPK/JNK/Akt Signaling Node That Regulates Obesity-Induced Insulin Resistance

Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high-fat diet-fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice, resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.

Compatibility of Tanshinone IIA and Astragaloside IV in attenuating hypoxia-induced cardiomyocytes injury

Ethnopharmacological relevanceHerbal medicines including Tanshinone IIA (TanIIA) and Astragaloside IV (AsIV) are widely used in Asia as therapeutic agents for cardiovascular diseases, due to their complementary roles and shared properties based on the theory of traditional Chinese medicine and pharmacological researches. However, the underlying pathological mechanisms for their efficacy are still unclear. In addition, the compatibility or incompatibility of the herbal medicines when administered with other herbal remedies or with prescription drugs is unknown. Aim of the studyWe aimed to investigate the compatibility of TanIIA and AsIV in protecting cardiomyocytes against hypoxia-induced injury. Materials and methodsCultured cardiomyocytes were stimulated in hypoxia condition, in the absence or presence of the two herbal compounds, TanIIA and AsIV. Indicators were determined by cytotoxicity assay, quantitative PCR, ELISA, flow cytometry assay, immunofluorescence staining and western blot. ResultsEither TanIIA alone or the combined herbal compounds inhibited hypoxia-triggered chemokines production including CCL2/5/19, CXCL2 and Transwell assay-indicated monocyte/macrophage recruitment, cytokines production including TNF-α and IL-6. While AsIV alone or the combined herbal compounds attenuated hypoxia-induced cell apoptosis indicated by decreased Annexin V+ cells and the ratio of Bax/Bcl-2, but no significant effect of the herbal compounds was observed in modulating cell apoptosis following both hypoxia and TNF-α stimulation. As an anti-apoptotic factor, stress granule formation was further enhanced by AsIV alone or the combined herbal compounds in hypoxia or heat shock stress. Moreover, immunoblotting analysis indicated that stress-responsive mitogen-activated protein kinases (MAPK) pathways including the phosphorylation of ERK1/2, p38 and JNK were inhibited while the phosphorylation of Akt in phosphatidylinositol 3-kinase (PI3K) -Akt pathway for cell survival was restored by the herbal compounds. Among these results, the combination of TanIIA and AsIV comprised most of the beneficial properties tested, although their combination did not improve the maximal effects achieved by any of the compounds alone. ConclusionsTaken together, these data suggest a compatibility of TanIIA and AsIV in protecting cardiomyocyte against hypoxia-induced injury.

Molecular characterization of biotic and abiotic stress-responsive MAP kinase genes, IbMPK3 and IbMPK6, in sweetpotato

Plants are continually exposed to numerous environmental stresses. To decrease damage caused by these potentially detrimental factors, various stress-related signaling cascades are activated in plants. One such stress-responsive signaling pathway, the mitogen-activated protein kinase (MAPK) module, plays a critical role in diverse plant stress responses. Here, we functionally characterized biotic and abiotic stress-responsive MAPK genes, IbMPK3 and IbMPK6, from sweetpotato. IbMPK3/6 contain totally 11 MAPK conserved subdomains and the phosphorylating motif TEY. Bacterially expressed IbMPK3/6 could be autophosphorylated in vitro, and these proteins phosphorylated universal kinase substrate, such as myelin basic protein. IbMPK3/6 transcripts were expressed in leaf, stem, and root of sweetpotato cultivars with storage roots of various colors. IbMPK3 and IbMPK6 were induced by various biotic/abiotic stress treatments. Furthermore, the kinase activity of IbMPK3/6 was induced during early NaCl, SA, H2O2, and ABA treatment. IbMPK3/6 were predominantly localized to the nucleus. To determine the biological functions of IbMPK3/6, we transiently expressed the IbMPK genes in tobacco (Nicotiana benthamiana) leaves, which resulted in enhanced tolerance to bacterial pathogen and increased expression of pathogenesis-related (PR) genes. These data demonstrate that IbMPK3 and IbMPK6 play significant roles in plant responses to environmental stress.

Morin Attenuates Ovalbumin-Induced Airway Inflammation by Modulating Oxidative Stress-Responsive MAPK Signaling.

Asthma is one of the most common inflammatory diseases characterized by airway hyperresponsiveness, inflammation, and remodeling. Morin, an active ingredient obtained from Moraceae plants, has been demonstrated to have promising anti-inflammatory activities in a range of disorders. However, its impacts on pulmonary diseases, particularly on asthma, have not been clarified. This study was designed to investigate whether morin alleviates airway inflammation in chronic asthma with an emphasis on oxidative stress modulation. In vivo, ovalbumin- (OVA-) sensitized mice were administered with morin or dexamethasone before challenge. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained to perform cell counts, histological analysis, and enzyme-linked immunosorbent assay. In vitro, human bronchial epithelial cells (BECs) were challenged by tumor necrosis factor alpha (TNF-α). The supernatant was collected for the detection of the proinflammatory proteins, and the cells were collected for reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) evaluations. Severe inflammatory responses and remodeling were observed in the airways of the OVA-sensitized mice. Treatment with morin dramatically attenuated the extensive trafficking of inflammatory cells into the BALF and inhibited their infiltration around the respiratory tracts and vessels. Morin administration also significantly suppressed goblet cell hyperplasia and collagen deposition/fibrosis and dose-dependently inhibited the OVA-induced increases in IgE, TNF-α, interleukin- (IL-) 4, IL-13, matrix metalloproteinase-9, and malondialdehyde. In human BECs challenged by TNF-α, the levels of proteins such as eotaxin-1, monocyte chemoattractant protein-1, IL-8 and intercellular adhesion molecule-1, were consistently significantly decreased by morin. Western blotting and the 2′,7′-dichlorofluorescein assay revealed that the increases in intracellular ROS and MAPK phosphorylation were abolished by morin, implying that ROS/MAPK signaling contributes to the relief of airway inflammation. Our findings indicate for the first time that morin alleviates airway inflammation in chronic asthma, which probably occurs via the oxidative stress-responsive MAPK pathway, highlighting a novel profile of morin as a potent agent for asthma management.

The mitogen-activated protein kinase CgHog1 is required for iron homeostasis, adherence and virulence in Candida glabrata.

Candida glabrata has emerged as a major fungal pathogen over the last two decades, although our understanding of its survival strategies inside the mammalian host remains rudimentary. An important requirement for survival in vivo is the ability to acquire critical nutrients such as iron from host niches of varied iron content. In the present study, we demonstrate for the first time that C. glabrata cells respond to high external iron levels via activation of two stress-responsive mitogen-activated protein kinases, CgHog1 and CgSlt2, and lack of either kinase results in sensitivity to the high-iron medium. Furthermore, we show that CgHOG1 deletion led to perturbed iron homeostasis (elevated intracellular iron content and high mitochondrial aconitase activity), reduced survival in macrophages and attenuated virulence in the murine model of disseminated candidiasis. Consistently, several genes implicated in iron acquisition and storage displayed deregulated expression in the Cghog1∆ mutant. Genome-wide transcriptional profiling analysis revealed upregulation of genes implicated in DNA repair, RNA processing and autophagy, and downregulation of genes related to cellular respiration and organonitrogen compound metabolism under iron-limiting conditions. In contrast, genes involved in the respiratory electron transport chain were induced under iron-replete conditions. Gene expression microarrays also identified a set of iron-responsive regulon in C. glabrata. Lastly, we present evidence for the iron-regulated expression of the major adhesin-encoding EPA1 gene, decreased histone deacetylase activity in a high-iron environment and increased adherence of iron-surplus-medium-grown C. glabrata cells to epithelial cells. Together, our findings yield novel insights into iron abundance-based regulation of transcriptional and mitogen-activated protein kinase signaling pathways in C. glabrata.

The role of three calcineurin subunits and a related transcription factor (Crz1) in conidiation, multistress tolerance and virulence in Beauveria bassiana.

The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16-38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in ΔcnB, which showed a large proportion of abnormally branched germlings but were less defective in ΔcnA1 and ΔcnA2. Conidiation defects also occurred in Δcrz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca(2+), Mn(2+), Zn(2+), Mg(2+), two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only ΔcnB and Δcrz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.

In silico identification and characterization of the MAPK family members of unicellular model eukaryote Tetrahymena thermophila

The biological function and evolutionary diversity of the mitogen-activated protein kinase (MAPK) family have mostly been studied in fungi, animals and plants, with very limited information from lower eukaryotes. This study aimed to describe the MAPKs of unicellular Tetrahymena thermophila. Eight members of the T. thermophila MAPK (TtMPK) gene family, in addition to previously reported TtMPK1, TtMPK2 and TtMPK3, were identified bioinformatically using a T. thermophila genome database. Phylogenetic analysis assigned the TtMPKs into two major groups, ERK1/2-like (TtMPK1, 2, 3, 5, 6, 7, 8, and 9) as stress-responsive MAPKs for biotic and abiotic stresses, and ERK7/8-like (TtMPK4, 10, and 11) as cell-cycle-associated protein kinases for biotic factors. Semi-quantitative RT-PCR analysis of the TtMPKs showed high mRNA expression at 30°C; however, only TtMPK5 and TtMPK6 showed high expression at 37°C. Osmotic shock by 100mM NaCl only increased the expression of TtMPK2, whereas 20mM NaCl reduced the expression of all MPKs to almost zero. The results suggested that T. thermophila MAPKs are among the closest representatives of the ancestors of the eukaryotic MAPK family. Although no functional characterization of MPKs was performed, this study is the first report of the genome-wide MAPK family in T. thermophila.

Arabidopsis Protein Phosphatase 2C ABI1 Interacts with Type I ACC Synthases and Is Involved in the Regulation of Ozone-Induced Ethylene Biosynthesis

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways.

Characterization and expression analysis of mitogen-activated protein kinase cascade genes in wheat subjected to phosphorus and nitrogen deprivation, high salinity, and drought

Mitogen-activated protein kinase (MAPK) cascades are conserved signal-transducing modules that have important functions in plant growth and development as well as in diverse biotic and abiotic stress responses. In this study, six MAP kinase kinase kinase genes, two MAP kinase kinase genes, and 11 MAP kinase genes in wheat were characterized. The MAPK cascade members of wheat were named based on their homologs in Brachypodium distachyon and Arabidopsis. The polypeptides translated from these MAPK cascade genes share conserved domains similar to those reported in B. distachyon and Arabidopsis, and such domains are involved in protein interaction and phosphorylation. Expression analysis results of the MAPK cascade members revealed that some of the selected genes were involved in responses to phosphorus (P; ten genes) and nitrogen (N; five genes) deprivation, salinity (five genes), and drought (three genes). Such genes were either upregulated or downregulated. Temporal expression profile analysis of the stress-responsive MAPK members indicated that all of these genes were progressively regulated by stress and exhibited typical recovery responses when these genes were exposed again to normal growth conditions. Two MAPK members, TaMPK6 and TaMPK16, responded to four stress factors, including deprivations of P and N as well as salt and drought stress. TaMPK4, TaMPK12;1, TaMPK14, and TaMPK17 were responsive to two stress factors. These results suggested that various MAPK cascade members in wheat are involved in mediating signal transductions by transcriptionally regulating P and N deprivations, high salinity, and drought. TaMPK6 and TaMPK16, together with TaMPK4, TaMPK12;1, TaMPK14, and TaMPK17 have potential functions in mediating plant responses and tolerance to two or more stress factors by distinct MAPK cascade modules. Our data have provided information about MAPK modules and put further insight into their biological functions in abiotic stress response of wheat.