Identification and analysis of MAPK cascade gene families of Camellia oleifera and their roles in response to cold stress.

BACKGROUND: Low-temperature severely limits the growth and development of Camellia oleifera (C. oleifera). The mitogen-activated protein kinase (MAPK) cascade plays a key role in the response to cold stress. METHODS AND RESULTS: Our study aims to identify MAPK cascade genes in C. oleifera and reveal their roles in response to cold stress. In our study, we systematically identified and analyzed the MAPK cascade gene families of C. oleifera, including their physical and chemical properties, conserved motifs, and multiple sequence alignments. In addition, we characterized the interacting networks of MAPKK kinase (MAPKKK)-MAPK kinase (MAPKK)-MAPK in C. oleifera. The molecular mechanism of cold stress resistance of MAPK cascade genes in wild C. oleifera was analyzed by differential gene expression and real-time quantitative reverse transcription-PCR (qRT-PCR). CONCLUSION: In this study, 21 MAPKs, 4 MAPKKs and 55 MAPKKKs genes were identified in the leaf transcriptome of C. oleifera. According to the phylogenetic results, MAPKs were divided into 4 groups (A, B, C and D), MAPKKs were divided into 3 groups (A, B and D), and MAPKKKs were divided into 2 groups (MEKK and Raf). Motif analysis showed that the motifs in each subfamily were conserved, and most of the motifs in the same subfamily were basically the same. The protein interaction network based on Arabidopsis thaliana (A. thaliana) homologs revealed that MAPK, MAPKK, and MAPKKK genes were widely involved in C. oleifera growth and development and in responses to biotic and abiotic stresses. Gene expression analysis revealed that the CoMAPKKK5/CoMAPKKK43/CoMAPKKK49-CoMAPKK4-CoMAPK8 module may play a key role in the cold stress resistance of wild C. oleifera at a high-elevation site in Lu Mountain (LSG). This study can facilitate the mining and utilization of genetic resources of C. oleifera with low-temperature tolerance.

Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

Biochanin A mitigates ulcerative colitis and intestinal inflammation in mice by inhibiting MAPK/NF-kB (p65) axis.

Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 µM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1ß (p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS) by assessing colon length, disease activity index (DAI) scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK) axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO) activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB (p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.

Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

Dimethyl ester of bilirubin ameliorates Naja naja snake venom-induced lung toxicity in mice via inhibiting NLRP3 inflammasome and MAPKs activation.

Naja naja snake bite causes thousands of deaths worldwide in a year. N. naja envenomed victims exhibit both local and systemic reactions that potentially lead to death. In clinical practice, pulmonary complications in N. naja envenomation are commonly encountered. However, the molecular mechanisms underlying N. naja venom-induced lung toxicity remain unknown. Here, we reasoned that N. naja venom-induced lung toxicity is prompted by NLRP3 inflammasome and MAPKs activation in mice. Treatment with dimethyl ester of bilirubin (BD1), significantly inhibited the N. naja venom-induced activation of NLRP3 inflammasome and MAPKs both in vivo and in vitro (p < 0.05). Further, BD1 reduced N. naja venom-induced recruitment of inflammatory cells, and hemorrhage in the lung toxicity examined by histopathology. BD1 also diminished N. naja venom-induced local toxicities in paw edema and myotoxicity in mice. Furthermore, BD1 was able to enhance the survival time against N. naja venom-induced mortality in mice. In conclusion, the present data showed that BD1 alleviated N. naja venom-induced lung toxicity by inhibiting NLRP3 inflammasome and MAPKs activation. Small molecule inhibitors that intervene in venom-induced toxicities may have therapeutic applications complementing anti-snake venom.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

StMAPK10 gene functional identification and analysis in drought resistance of potato crop (Solanum tuberosum L.).

All over the world, potato (Solanum tuberosum L.) production is constrained by several biotic and abiotic factors. Many techniques and mechanisms have been used to overcome these hurdles and increase food for the rising population. In crop plants, the mitogen-activated protein kinase (MAPK) cascade, a significant regulator of the MAPK pathway under various biotic and abiotic stress conditions, is one of the targets to increase productivity. MAPK plays a significant role under drought stress in potato. However, the function of MAPK in drought resistance in potato is poorly understood. In this study, we wanted to identify the function of StMAPK10 in the drought resistance in potato. StMAPK10 was up-regulated under drought conditions and dynamically modulated by abiotic stresses. Over-expression and down-regulation of StMAPK10 revealed that StMAPK10 stimulated potato growth under drought conditions, as demonstrated by changes in SOD, CAT, and POD activity, as well as H2O2, proline, and MDA content. StMAPK10 up-regulation exaggerated the drought resistance of the potato plant by uplifting antioxidant activities and photosynthetic indices. Overexpressed-StMAPK10 potato lines showed highly significant results for physiological and photosynthetic indices in response to drought stress, while knockdown expression showed opposite outcomes. Additionally, subcellular localization and phenotypic analysis of transgenic and non-transgenic plants substantiated the role of the increased expression of StMAPK10 against drought stress. The results could provide novel insights into the functionality of StMAPK10 in drought responses and conceivable mechanisms.

Off-target effect of high-dose sildenafil on adenosine 5'- diphosphate and collagen-induced platelet activation through mitogen-activated protein kinase pathway in treated BALB/C mice and in vitro experiments: A preliminary study.

ABSTRACT: Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and sudden cardiac death in a few case reports. Therefore, we investigated the in vitro and in vivo effect of sildenafil treatment and dosage on platelet activation and mitogen-activated protein kinase (MAPK) phosphorylation. BALB/C mice were segregated into four groups, each having four mice each (control, low [3.25 mg/kg], medium [6.5 mg/kg], and high [13 mg/kg] sildenafil), and after the treatment, blood was drawn from each mouse and washed platelets prepared. Washed platelets were incubated with CD41 PE-Cy7 and Phospho-p38 MAPK PE antibodies and analyzed using a flow cytometer for platelet activation and adenosine 5'- diphosphate (ADP)/collagen-induced MAPK phosphorylation. Washed platelets obtained from the venous blood of 18 human volunteers, were incubated with PAC-1 FITC and Phospho-p38 MAPK PE antibodies, and platelet activation (ADP and collagen), followed by flow cytometry analysis. There was a significant increase in both platelet activation as well as MAPK phosphorylation in the presence of collagen in the high-dose (13 mg/kg) sildenafil group (P = 0.000774). Further, increased platelet activation was observed in samples that were treated with high-dose sildenafil as compared to the untreated samples (P < 0.00001). These studies show the risk of prothrombotic episodes in patients on high-dose sildenafil (100 mg), in those with even mild endothelial dysfunction due to ADP, and collagen-induced platelet activation through MAPK phosphorylation, which was not seen in the low-and intermediate-dose cohorts.

The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

Retracted: MD-1 Deficiency Accelerates Myocardial Inflammation and Apoptosis in Doxorubicin-Induced Cardiotoxicity by Activating the TLR4/MAPKs/Nuclear Factor kappa B (NF-κB) Signaling Pathway.

This publication has been retracted by the Editor due to the identification of non-original figure images and manuscript content that raise concerns regarding the credibility and originality of the study and the manuscript. Reference: Ying-Jun Zhang, He Huang, Yu Liu, Bin Kong, Guangji Wang. MD-1 Deficiency Accelerates Myocardial Inflammation and Apoptosis in Doxorubicin-Induced Cardiotoxicity by Activating the TLR4/MAPKs/Nuclear Factor kappa B (NF-kappaB) Signaling Pathway. Med Sci Monit, 2019; 25: 7898-7907. DOI: 10.12659/MSM.919861.

Green tea extract suppresses airway inflammation via oxidative stress-driven MAPKs/MMP-9 signaling in asthmatic mice and human airway epithelial cells.

Introduction: The anti-inflammatory effect of green tea extract (GTE) has been confirmed in asthmatic mice, however, the pharmacological mechanism is not fully elucidated. Methods: To investigate the therapeutic efficacy of GTE in asthma and identify specific pathways, murine model of allergic asthma was established by ovalbumin (OVA) sensitization and the challenge for 4 weeks, with oral treatment using GTE and dexamethasone (DEX). Inflammatory cell counts, cytokines, OVA-specific IgE, airway hyperreactivity, and antioxidant markers in the lung were evaluated. Also, pulmonary histopathological analysis and western blotting were performed. In vitro, we established the model by stimulating the human airway epithelial cell line NCI-H292 using lipopolysaccharide, and treating with GTE and mitogen-activated protein kinases (MAPKs) inhibitors. Results: The GTE100 and GTE400 groups showed a decrease in airway hyperresponsiveness and the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) compared to the OVA group. GTE treatment also reduced interleukin (IL)-13, IL-5, and IL-4 levels in the BALF, and OVA-specific immunoglobulin E levels in the serum compared to those in the OVA group. GTE treatment decreased OVA-induced mucus secretion and airway inflammation. In addition, GTE suppressed the oxidative stress, and phosphorylation of MAPKs, which generally occurs after exposure to OVA. GTE administration also reduced matrix metalloproteinase-9 activity and protein levels. Conclusion: GTE effectively inhibited asthmatic respiratory inflammation and mucus hyperproduction induced by OVA inhalation. These results suggest that GTE has the potential to be used for the treatment of asthma.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Design, synthesis and molecular modeling of novel D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives as anti-inflammatory agents by inhibition of COX-2/iNOS production and down-regulation of NF-κB/MAPKs in LPS-induced RAW264.7 macrophage cells.

It has been reported that 4,5-dihydropyrazole and thiazole derivatives have many biological functions, especially in the aspect of anti-inflammation. According to the strategy of pharmacophore combination, we introduced thiazolinone and dihydropyrazole moiety into steroid skeleton to design and synthesize a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives, and assessed their in vitro anti-inflammatory profiles against Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. The anti-inflammatory activities assay demonstrated that compound 12e was considered as the most effective anti-inflammatory drug, which suppressed the expression of pro-inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), it also dose-dependently inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 macrophage cells. Furthermore, the results of the Western blot analysis showed a correlation between the inhibition of the Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) signaling pathways and the suppressive effects of compound 12e on pro-inflammatory cytokines. Molecular docking studies of compound 12e into the COX-2 protein receptor (PDB ID: 5IKQ) active site was performed to rationalize their COX-2 inhibitory potency. The results were found to be in line with the biological findings as they exerted more favorable interactions compared to that of dexamethasone (DXM), explaining their remarkable COX-2 inhibitory activity. The findings revealed that these candidates could be identified as potent anti-inflammatory agents, compound 12e could be a promising drug for the treatment of inflammatory diseases.

The Hog1 MAPK substrate governs Candida glabrata-epithelial cell adhesion via the histone H2A variant.

CgHog1, terminal kinase of the high-osmolarity glycerol signalling pathway, orchestrates cellular response to multiple external stimuli including surplus-environmental iron in the human fungal pathogen Candida glabrata (Cg). However, CgHog1 substrates remain unidentified. Here, we show that CgHog1 adversely affects Cg adherence to host stomach and kidney epithelial cells in vitro, but promotes Cg survival in the iron-rich gastrointestinal tract niche. Further, CgHog1 interactome and in vitro phosphorylation analysis revealed CgSub2 (putative RNA helicase) to be a CgHog1 substrate, with CgSub2 also governing iron homeostasis and host adhesion. CgSub2 positively regulated EPA1 (encodes a major adhesin) expression and host adherence via its interactor CgHtz1 (histone H2A variant). Notably, both CgHog1 and surplus environmental iron had a negative impact on CgSub2-CgHtz1 interaction, with CgHTZ1 or CgSUB2 deletion reversing the elevated adherence of Cghog1Δ to epithelial cells. Finally, the surplus-extracellular iron led to CgHog1 activation, increased CgSub2 phosphorylation, elevated CgSub2-CgHta (canonical histone H2A) interaction, and EPA1 transcriptional activation, thereby underscoring the iron-responsive, CgHog1-induced exchange of histone partners of CgSub2. Altogether, our work mechanistically defines how CgHog1 couples Epa1 adhesin expression with iron abundance, and point towards specific chromatin composition modification programs that probably aid fungal pathogens align their adherence to iron-rich (gut) and iron-poor (blood) host niches.

Deregulated JNK signaling enhances apoptosis during hyperthermia.

PURPOSE: c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. METHODS AND MATERIALS: The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. RESULTS: Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1-SEK1-JNK pathway and apoptosis. CONCLUSION: JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1-SEK1-JNK pathway and subsequent apoptosis.

Resveratrol attenuates cyclosporin A-induced upregulation of the thromboxane A2 receptor and hypertension via the AMPK/SIRT1 and MAPK/NF-κB pathways in the rat mesenteric artery.

Cyclosporin A, an immunosuppressive agent, is extensively utilized for the prevention of transplant rejection and treat autoimmune disease in the clinic, despite its association with a high risk of hypertension development among patients. Resveratrol is a kind of non-flavonoid phenolic compound that widely exists in many plants. The aim of the present study was to investigate the mechanism by which resveratrol ameliorates cyclosporin A-induced hypertension. The arterial rings of the mesentery were incubated with cyclosporin A and resveratrol in vitro. Rats were administered cyclosporin A and/or resveratrol for 3 weeks in vivo. Blood pressure was measured via the tail arteries. Vasoconstriction curves were recorded using a sensitive myograph. The protein expression was evaluated through Western blotting. This study demonstrated that resveratrol mitigated the cyclosporin A-induced increase in blood pressure in rats. Furthermore, resveratrol markedly inhibited the cyclosporin A-induced upregulation of thromboxane A2 receptor-mediated vasoconstriction in the rat mesenteric artery both in vitro and in vivo. Moreover, resveratrol activated AMPK/SIRT1 and inhibited the MAPK/NF-κB signaling pathway. In conclusion, resveratrol restored the cyclosporin A-induced upregulation of the thromboxane A2 receptor and hypertension via the AMPK/SIRT1 and MAPK/NF-κB pathways in rats.

Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

Xbra modulates the activity of linker region phosphorylated Smad1 during Xenopus development.

The Bmp/Smad1 pathway plays a crucial role in developmental processes and tissue homeostasis. Mitogen-activated protein kinase (Mapk)/Erk mediated phosphorylation of Smad1 in the linker region leads to Smad1 degradation, cytoplasmic retention and inhibition of Bmp/Smad1 signaling. While Fgf/Erk pathway has been documented to inhibit Bmp/Smad1 signaling, several studies also suggests the cooperative interaction between these two pathways in different context. However, the precise role and molecular pathway of this collaborative interaction remain obscure. Here, we identified Xbra induced by Fgf/Erk signaling as a factor in a protective mechanism for Smad1. Xbra physically interacted with the linker region phosphorylated Smad1 to make Xbra/Smad1/Smad4 trimeric complex, leading to Smad1 nuclear localization and protecting it from ubiquitin-mediated proteasomal degradation. This interaction of Xbra/Smad1/Smad4 led to sustained nuclear localization of Smad1 and the upregulation of lateral mesoderm genes, while concurrently suppression of neural and blood forming genes. Taken together, the results suggests Xbra-dependent cooperative interplays between Fgf/Erk and Bmp/Smad1 signaling during lateral mesoderm specification in Xenopus embryos.

Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae.

Mitogen-activated protein kinase (MAPK) pathways control the response to intrinsic and extrinsic stimuli. In the budding yeast Saccharomyces cerevisiae, cells undergo filamentous growth, which is regulated by the fMAPK pathway. To better understand the regulation of the fMAPK pathway, a genetic screen was performed to identify spontaneous mutants with elevated activity of an fMAPK pathway-dependent growth reporter (ste4  FUS1-HIS3). In total, 159 mutants were isolated and analyzed by secondary screens for invasive growth by the plate-washing assay and filament formation by microscopy. Thirty-two mutants were selected for whole-genome sequencing, which identified new alleles in genes encoding known regulators of the fMAPK pathway. These included gain-of-function alleles in STE11, which encodes the MAPKKK, as well as loss-of-function alleles in KSS1, which encodes the MAP kinase, and loss-of-function alleles in RGA1, which encodes a GTPase-activating protein (GAP) for CDC42. New alleles in previously identified pathway modulators were also uncovered in ALY1, AIM44, RCK2, IRA2, REG1, and in genes that regulate protein folding (KAR2), glycosylation (MNN4), and turnover (BLM10). Mutations leading to C-terminal truncations in the transcription factor Ste12p were also uncovered that resulted in elevated reporter activity, identifying an inhibitory domain of the protein from residues 491 to 688. We also find that a diversity of filamentous growth phenotypes can result from combinatorial effects of multiple mutations and by loss of different regulators of the response. The alleles identified here expand the connections surrounding MAPK pathway regulation and reveal new features of proteins that function in the signaling cascade.

A Method to Conditionally Measure Target Engagement at Intracellular RAS and RAF Complexes.

Dysfunction of the RAS/mitogen-activated protein kinase (MAPK) pathway is a common driver of human cancers. As such, both the master regulator of the pathway, RAS, and its proximal kinase effectors, RAFs, have been of interest as drug targets for decades. Importantly, signaling within the RAS/MAPK pathway is highly coordinated due to the formation of a higher-order complex called the RAS/RAF signalosome, which may minimally contain dimers of both RAS and RAF protomers. In the disease state, RAS and RAF assemble in homo- and/or heterodimeric forms. Traditionally, drug development campaigns for both RAS and RAF have utilized biochemical assays of purified recombinant protein. As these assays do not query the RAS or RAF proteins in their full-length and complexed forms in cells, potency results collected using these assays have often failed to correlate with inhibition of the MAPK pathway. To more accurately quantify engagement at this signaling components, we present a bioluminescence resonance energy transfer (BRET)-based method to conditionally measure target engagement at individual protomers within the RAS/RAF signalosome in live cells.