Protein Kinase Research Articles

L-Fucose-Rich Sulfated Glycans from Edible Brown Seaweed: A Promising Functional Food for Obesity and Energy Expenditure Improvement.

The global obesity epidemic, exacerbated by the sedentary lifestyle fostered by the COVID-19 pandemic, presents a growing socioeconomic burden due to decreased physical activity and increased morbidity. Current obesity treatments show promise, but they often come with expensive medications, frequent injections, and potential side effects, with limited success in improving obesity through increased energy expenditure. This study explores the potential of a refined sulfated polysaccharide (SPSL), derived from the brown seaweed Scytosiphon lomentaria (SL), as a safe and effective anti-obesity treatment by promoting energy expenditure. Chemical characterization revealed that SPSL, rich in sulfate and L-fucose content, comprises nine distinct sulfated glycan structures. In vitro analysis demonstrated potent anti-lipogenic properties in adipocytes, mediated by the downregulation of key adipogenic modulators, including 5' adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ (PPARγ) pathways. Inhibiting AMPK attenuated the anti-adipogenic effects of SPSL, confirming its involvement in the mechanism of action. Furthermore, in vivo studies using zebrafish models showed that SPSL increased energy expenditure and reduced lipid accumulation. These findings collectively highlight the therapeutic potential of SPSL as a functional food ingredient for mitigating obesity-related metabolic dysregulation by promoting energy expenditure. Further mechanistic and preclinical investigations are warranted to fully elucidate its mode of action and evaluate its efficacy in obesity management, potentially offering a novel, natural therapeutic avenue for this global health concern.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

EGFR and EGFRvIII coopt host defense pathways, promoting progression in glioblastoma.

Co-amplification of EGFR and EGFRvIII, a tumor-specific truncation mutant of EGFR, represent hallmark genetic lesions in glioblastoma. We used phospho-proteomics, RNA-sequencing, TCGA data and glioblastoma cell culture and mouse models to study the signal transduction mediated by EGFR and EGFRvIII. We report that EGFR and EGFRvIII stimulate the innate immune defense receptor Toll-like Receptor 2 (TLR2); and that knockout of TLR2 dramatically improved survival in orthotopic glioblastoma xenografts. EGFR and EGFRvIII activated TLR2 in a ligand-independent manner, promoting tumor growth and immune evasion. We show that EGFR and EGFRvIII cooperate to activate the Rho-associated protein kinase ROCK2, which modulated malignant progression both by activating TLR2 and WNT signaling, and through remodeling the tumor microenvironment. Together, our findings show that EGFR and EGFRvIII cooperate to drive tumor progression through ROCK2 and downstream WNT-β-catenin/TLR2 signaling pathways.

Mangiferin: An effective agent against human malignancies

AbstractMangiferin is a bioactive substance present in high concentration in mangoes and also in some other fruits. Owing to its potential as a chemopreventive and chemotherapeutic agent against several types of cancer, this unique, significant, and well‐researched polyphenol has received a lot of attention recently. It possesses the ability to treat cancers, including rectal cancer, prostate cancer, ovarian cancer, leukemia, gastric cancer, liver cancer, chronic pancreatitis, and lung cancer. It can control/regulate multiple key signaling pathways, such as signal transducer and activator of transcription 3 (STAT3), second mitochondria‐derived activator of caspases/direct inhibitor of apoptosis (IAP)‐binding protein with low propidium iodide (pl) (Smac/DIABLO) nuclear factor kappa B (NF‐κB), phosphatidylinositol 3 kinase/protein 3 kinase (PI3K/Akt), transforming growth factor beta/suppressor of mothers against decapentaplegic (TGF‐β/SMAD), c‐jun N‐terminal kinase/p38 mitogen‐activated protein kinase (JNK/p38‐MAPK), and phosphor‐I kappa B kinase (p‐IκB), which are crucial to the development of cancers. By triggering apoptotic signals and halting the advancement of the cell cycle, it can also prevent some cancer cell types from proliferating and developing. It has been revealed that mangiferin targets a variety of adhesion molecules, cytokines, pro‐inflammatory transcription factors, kinases, chemokines, growth factors, and cell‐cycle proteins. By means of preventing the onset, advancement, and metastasis of cancer, these targets may mediate the chemopreventive and therapeutic effects of mangiferin. Mangiferin has confirmed potential benefits in lung, cervical, breast, brain, and prostate cancers as well as leukemia whether administered alone or in combination with recognized anticancer compounds. More clinical trials and research investigations are required to completely unleash the potential of mangiferin, which may lower the risk of cancer onset and act as a preventive and therapeutic alternative for a number of cancers.

Microglial morphological/inflammatory phenotypes and endocannabinoid signaling in a preclinical model of periodontitis and depression

BackgroundDepression is a chronic psychiatric disease of multifactorial etiology, and its pathophysiology is not fully understood. Stress and other chronic inflammatory pathologies are shared risk factors for psychiatric diseases, and comorbidities are features of major depression. Epidemiological evidence suggests that periodontitis, as a source of low-grade chronic systemic inflammation, may be associated with depression, but the underlying mechanisms are not well understood.MethodsPeriodontitis (P) was induced in Wistar: Han rats through oral gavage with the pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum for 12 weeks, followed by 3 weeks of chronic mild stress (CMS) to induce depressive-like behavior. The following four groups were established (n = 12 rats/group): periodontitis and CMS (P + CMS+), periodontitis without CMS, CMS without periodontitis, and control. The morphology and inflammatory phenotype of microglia in the frontal cortex (FC) were studied using immunofluorescence and bioinformatics tools. The endocannabinoid (EC) signaling and proteins related to synaptic plasticity were analyzed in FC samples using biochemical and immunohistochemical techniques.ResultsUltrastructural and fractal analyses of FC revealed a significant increase in the complexity and heterogeneity of Iba1 + parenchymal microglia in the combined experimental model (P + CMS+) and increased expression of the proinflammatory marker inducible nitric oxide synthase (iNOS), while there were no changes in the expression of cannabinoid receptor 2 (CB2). In the FC protein extracts of the P + CMS + animals, there was a decrease in the levels of the EC metabolic enzymes N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) compared to those in the controls, which extended to protein expression in neurons and in FC extracts of cannabinoid receptor 1 (CB1) and to the intracellular signaling molecules phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2). The protein levels of brain-derived neurotrophic factor (BDNF) and synaptophysin were also lower in P + CMS + animals than in controls.ConclusionsThe combined effects on microglial morphology and inflammatory phenotype, the EC signaling, and proteins related to synaptic plasticity in P + CMS + animals may represent relevant mechanisms explaining the association between periodontitis and depression. These findings highlight potential therapeutic targets that warrant further investigation.Graphical

Patient-derived acellular ascites fluid affects drug responses in ovarian cancer cell lines through the activation of key signalling pathways.

Malignant ascites is commonly produced in advanced epithelial ovarian cancer (EOC) and serves as unique microenvironment for tumour cells. Acellular ascites fluid (AAF) is rich in signalling molecules and has been proposed to play a role in the induction of chemoresistance. Through in vitro testing of drug sensitivity and by assessing intracellular phosphorylation status in response to mono- and combination treatment of five EOC cell lines after incubation with AAFs derived from 20 different patients, we investigated the chemoresistance-inducing potential of ascites. We show that the addition of AAFs to the culture media of EOC cell lines has the potential to induce resistance to standard-of-care drugs (SCDs). We also show that AAFs induce time- and concentration-dependent activation of downstream signalling to signal transducer and activator of transcription 3 (STAT3), and concomitantly altered phosphorylation of mitogen-activated protein kinase kinase (MEK), phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) and nuclear factor NF-kappa-B (NFκB). Antibodies targeting the interleukin-6 receptor (IL6R) effectively blocked phosphorylation of STAT3 and STAT1. Treatments with SCDs were effective in reducing cell viability in only a third of 30 clinically relevant conditions examined, defined as combinations of drugs, different cell lines and AAFs. Combinations of SCDs and novel therapeutics such as trametinib, fludarabine or rapamycin were superior in another third. Notably, we could nominate effective treatment combinations in almost all conditions except in 4 out of 30 conditions, in which trametinib or fludarabine showed higher efficacy alone. Taken together, our study underscores the importance of the molecular characterisation of individual patients' AAFs and the impact on treatment resistance as providing clinically meaningful information for future precision treatment approaches in EOC.

8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway.

8-Prenylgenistein (8PG), a genistein derivative, is present in fermented soybeans (Glycine max), including cheonggukjang (CGJ), and exhibits osteoprotective, osteogenic, and antiadipogenic properties. However, the hepatoprotective effects of 8PG and its underlying molecular mechanisms remain largely unexplored. Here, we identified the high binding affinity of 8PG with AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), which acts as a potent AMPK activator that counteracts hepatic steatosis. Notably, 8PG exhibited better pharmacokinetics with greater absorption and higher plasma binding than the positive controls for the target proteins. Moreover, 8PG exerted non-carcinogenic activity in rats and significantly increased AMPK phosphorylation. Compound C, an AMPK inhibitor, did not antagonize 8PG-activated AMPK in HepG2 cells. 8PG significantly attenuated palmitate-induced lipid accumulation and enhanced phosphorylated AMPK and its downstream target, acetyl-CoA carboxylase. Further, 8PG activated nuclear SIRT1 at the protein level, which promoted fatty acid oxidation in palmitate-treated HepG2 cells. Overall, 8PG acts as a potent AMPK activator, further attenuating hepatic steatosis via the SIRT1-mediated pathway and providing new avenues for dietary interventions to treat metabolic dysfunction-associated steatotic liver disease (MASLD).

Glucose-Dependent Insulinotropic Polypeptide Inhibits AGE-Induced NADPH Oxidase-Derived Oxidative Stress Generation and Foam Cell Formation in Macrophages Partly via AMPK Activation.

Glucose-dependent insulinotropic polypeptide (GIP) of the incretin group has been shown to exert pleiotropic actions. There is growing evidence that advanced glycation end products (AGEs), senescent macromolecules formed at an accelerated rate under chronic hyperglycemic conditions, play a role in the pathogenesis of atherosclerotic cardiovascular disease in diabetes. However, whether and how GIP could inhibit the AGE-induced foam cell formation of macrophages, an initial step of atherosclerosis remains to be elucidated. In this study, we address these issues. We found that AGEs increased oxidized low-density-lipoprotein uptake into reactive oxygen species (ROS) generation and Cdk5 and CD36 gene expressions in human U937 macrophages, all of which were significantly blocked by [D-Ala2]GIP(1-42) or an inhibitor of NADPH oxidase activity. An inhibitor of AMP-activated protein kinase (AMPK) attenuated all of the beneficial effects of [D-Ala2]GIP(1-42) on AGE-exposed U937 macrophages, whereas an activator of AMPK mimicked the effects of [D-Ala2]GIP(1-42) on foam cell formation, ROS generation, and Cdk5 and CD36 gene expressions in macrophages. The present study suggests that [D-Ala2]GIP(1-42) could inhibit the AGE-RAGE-induced, NADPH oxidase-derived oxidative stress generation in U937 macrophages via AMPK activation and subsequently suppress macrophage foam cell formation by reducing the Cdk5-CD36 pathway.

Integration of Transcriptomics and WGCNA to Characterize Trichoderma harzianum-Induced Systemic Resistance in Astragalus mongholicus for Defense against Fusarium solani

Beneficial fungi of the genus Trichoderma are among the most widespread biocontrol agents that induce a plant’s defense response against pathogens. Fusarium solani is one of the main pathogens that can negatively affect Astragalus mongholicus production and quality. To investigate the impact of Trichoderma harzianum on Astragalus mongholicus defense responses to Fusarium solani, A. mongholicus roots under T. harzianum + F. solani (T + F) treatment and F. solani (F) treatment were sampled and subjected to transcriptomic analysis. A differential expression analysis revealed that 6361 differentially expressed genes (DEGs) responded to T. harzianum induction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 6361 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant–pathogen interaction pathway, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction pathway. Pathway analysis revealed that the PR1, formononetin biosynthesis, biochanin A biosynthesis, and CHIB, ROS production, and HSP90 may be upregulated by T. harzianum and play important roles in disease resistance. Our study further revealed that the H2O2 content was significantly increased by T. harzianum induction. Formononetin and biochanin A had the potential to suppress F. solani. Weighted gene coexpression network analysis (WGCNA) revealed one module, including 58 DEGs associated with T. harzianum induction. One core hub gene, RPS25, was found to be upregulated by T. harzianum, SA (salicylic acid) and ETH (ethephon). Overall, our data indicate that T. harzianum can induce induced systemic resistance (ISR) and systemic acquired resistance (SAR) in A. mongholicus. The results of this study lay a foundation for a further understanding of the molecular mechanism by which T. harzianum induces resistance in A. mongholicus.

MiR-509-3p promotes gastric cancer development by activating FOXM1-mediated p38/MK2 pathway

Gastric cancer (GC), a malignant tumor, is highly prevalent, particularly in Asia. miR-509-3p plays a crucial role in regulating tumorigenesis, but its mechanism in GC remains unclear. Potential targets of miR-509-3p were identified through database analysis (miRWalk, TargetScan, ENCORI, and TCGA). The binding site between miR-509-3p and forkhead box protein M1 (FOXM1) was confirmed using a dual-luciferase assay. CCK-8, EdU, Transwell, wound healing assays, flow cytometry, and Western blot analysis were employed to examine changes in proliferation, migration, invasion, apoptosis, FOXM1, and the p38 MAPK (p38)/MAPK-activated protein kinase 2 (MK2) pathway in GC cells (MNK-45 and HGC-27) after miR-509-3p overexpression or knockdown, FOXM1 overexpression, and application of the p38 pathway agonist Anisomycin. The size and weight of subcutaneous xenografts were measured, and the effects of miR-509-3p overexpression were analyzed through histopathological staining (Tunel immunofluorescence, HE staining, Ki67, and FOXM1 immunohistochemistry). The results showed that overexpression of miR-509-3p suppressed proliferation, migration, and invasion while accelerating apoptosis. Knockdown of miR-509-3p promoted malignant progression. miR-509-3p inhibited GC by regulating FOXM1-mediated p38/MK2 pathway activation, and miR-509-3p mimics restrained tumor growth in vivo through this pathway. In conclusion, miR-509-3p suppresses GC malignant progression by regulating FOXM1-mediated p38/MK2 pathway activation.

J147 treatment protects against traumatic brain injury by inhibiting neuronal endoplasmic reticulum stress potentially via the AMPK/SREBP-1 pathway

Endoplasmic reticulum (ER) stress is recognized as a crucial contributor to the progression of traumatic brain injury (TBI) and represents a potential target for therapeutic intervention. This study aimed to assess the potential of J147, a novel neurotrophic compound, in alleviating ER stress by modulating related signaling pathways, thereby promoting functional recovery in TBI. To this end, adult mice underwent controlled cortical impact (CCI) injury to induce TBI, followed by oral administration of J147 one-hour post-injury, with daily dosing for 3 to 7 days. Multiple behavioral assessments were conducted over 35 days, revealing a significant, dose-dependent improvement in neurofunctional recovery with J147 treatment. The neuropathological analysis demonstrated reduced acute neurodegeneration (observed at three days through FJC staining), enhanced long-term neuron survival (H&E and Nissl staining), and improved neuroplasticity (Golgi staining) at 35 days post-TBI. At the molecular level, TBIinduced AMP-activated protein kinase (AMPK) dephosphorylation, sterol regulatory element binding protein-1 (SREBP-1) activation, and upregulation of ER stress marker proteins, including phosphorylated eukaryotic initiation factor-2α (p-eIF2a), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in perilesional cortex neurons at three days post-injury. Notably, the J147 treatment significantly attenuated AMPK dephosphorylation, SERBP-1 activation, and expression of the ER stress markers. In summary, this study reveals the therapeutic promise of J147 in mitigating secondary brain damage associated with TBI and improving long-term functional recovery by modulating ER stress pathways.

Gut microbiota-derived Metabolite, Shikimic Acid, inhibits vascular smooth muscle cell proliferation and migration

Gut microbiota dysbiosis is linked to vascular wall disease, but the mechanisms by which gut microbiota cross-talk with the host vascular cells remain largely unknown. Shikimic acid (SA) is a biochemical intermediate synthesized in plants and microorganisms, but not mammals. Surprisingly, recent metabolomic profiling data demonstrate that SA is detectable in human and murine blood. In this study, analyzing data from germ-free rats, we provide evidence in support of SA as a bona fide gut microbiota-derived metabolite, emphasizing its biological relevance. Since vascular cells are the first cells exposed to circulating metabolites, in this study, we examined, for the first time, the effects and potential underlying molecular mechanisms of SA on vascular smooth muscle cell (VSMC) proliferation and migration, which play a key role in occlusive vascular diseases, such as post-angioplasty restenosis and atherosclerosis. We found that SA inhibits the proliferation and migration of human coronary artery SMCs. At the molecular level, unexpectedly, we found that SA activates, rather than inhibits, multiple pro-mitogenic signaling pathways in VSMCs, such as ERK1/2, AKT, and mTOR/p70S6K. Conversely, we found that SA activates the anti-proliferative AMP-activated protein kinase (AMPK) in VSMCs, a key cellular energy sensor and regulator. However, loss-of-function experiments demonstrate that AMPK does not mediate the inhibitory effects of SA on VSMC proliferation. In conclusion, these studies demonstrate that a microbiota-derived metabolite, SA, inhibits VSMC proliferation and migration in vitro and prompt further evaluation of the possible underlying molecular mechanisms and the potential protective role in VSMC-related vascular wall disease in vivo.

Low concentrations of the food contaminant Deoxynivalenol trigger apoptosis and impair GnRH-induced LH secretion in pituitary gonadotrope-like cells.

AbstractTheFusariummycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. Nearly all individuals are exposed to DON, due to it widespread presence in grains and grain-based products. Chronic DON poisoning is associated with growth retardation, immunotoxicity as well as impaired reproduction and fetal development. At the molecular level, DON alters intracellular signaling by activating mitogen-activated protein kinases (MAPKs) that modulate cell growth, differentiation, and apoptosis. Of note, these MAPKs are also critical mediators of gonadotrophin-releasing hormone (GnRH)-induced synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by pituitary gonadotrope cells. So far, no research has explored the potential endocrine-disrupting effects of DON on pituitary gonadotropins production. Herein, we show the first evidence that DON affects LH production by the immortalized gonadotrope-like cell line LβT2 in a concentration-dependent manner. Taken together, our experiments demonstrated that low concentrations of DON affect GnRH-induced signaling through a mechanism that, at least in part, involves apoptosis and inhibition of GnRH-induced phosphorylation of ERK-MAPK. Consequently, DON also affects the GnRH-induced expression ofCgaandLhb, two critical genes for LH synthesis and secretion by gonadotrope cells in mammals. This research broadens our knowledge of the toxicity of DON and brings a new depth to the potential neuroendocrine implications for reproduction.Graphical abstract

RNA-Seq and WGCNA Analyses Reveal Key Regulatory Modules and Genes for Salt Tolerance in Cotton

The problem of soil salinization has seriously hindered agricultural development. Cotton is a pioneering salinity-tolerant crop, so harvesting its key salinity-tolerant genes is important for improving crop salt tolerance. In this study, we analyzed changes in the transcriptome expression profiles of the salt-tolerant cultivar Lu Mian 28 (LM) and the salt-sensitive cultivar Zhong Mian Suo 12 (ZMS) after applying salt stress, and we constructed weighted gene co-expression networks (WGCNA). The results indicated that photosynthesis, amino acid biosynthesis, membrane lipid remodeling, autophagy, and ROS scavenging are key pathways in the salt stress response. Plant–pathogen interactions, plant hormone signal transduction, the mitogen-activated protein kinase (MAPK) signaling pathway, and carotenoid biosynthesis are the regulatory networks associated with these metabolic pathways that confer cotton salt tolerance. The gene-weighted co-expression network was used to screen four modules closely related to traits, identifying 114 transcription factors, including WRKYs, ERFs, NACs, bHLHs, bZIPs, and MYBs, and 11 hub genes. This study provides a reference for acquiring salt-tolerant cotton and abundant genetic resources for molecular breeding.

Mitogen-activated protein kinase signalling in rat hearts during postnatal development: MAPKs, MAP3Ks, MAP4Ks and DUSPs

Mammalian cardiomyocytes become terminally-differentiated during the perinatal period. In rodents, cytokinesis ceases after a final division cycle immediately after birth. Nuclear division continues and most cardiomyocytes become binucleated by ∼11 days. Subsequent growth results from an increase in cardiomyocyte size. The mechanisms involved remain under investigation. Mitogen-activated protein kinases (MAPKs) regulate cell growth/death: extracellular signal-regulated kinases 1/2 (ERK1/2) promote proliferation, whilst c-Jun N-terminal kinases (JNKs) and p38-MAPKs respond to cellular stresses. We assessed their regulation in rat hearts during postnatal development (2, 7, 14, and 28 days, 12 weeks) during which time there was rapid, substantial downregulation of mitosis/cytokinesis genes (Cenpa/e/f, Aurkb, Anln, Cdca8, Orc6) with lesser downregulation of DNA replication genes (Orcs1–5, Mcms2–7). MAPK activation was assessed by immunoblotting for total and phosphorylated (activated) kinases. Total ERK1/2 was downregulated, but not JNKs or p38-MAPKs, whilst phosphorylation of all MAPKs increased relative to total protein albeit transiently for JNKs. These profiles differed from activation of Akt (also involved in cardiomyocyte growth). Dual-specificity phosphatases, upstream MAPK kinase kinases (MAP3Ks), and MAP3K kinases (MAP4Ks) identified in neonatal rat cardiomyocytes by RNASeq were differentially regulated during postnatal cardiac development. The MAP3Ks that we could assess by immunoblotting (RAF kinases and Map3k3) showed greater downregulation of the protein than mRNA. MAP3K2/MAP3K3/MAP4K5 were upregulated in human failing heart samples and may be part of the “foetal gene programme” of re-expressed genes in disease. Thus, MAPKs, along with kinases and phosphatases that regulate them, potentially play a significant role in postnatal remodelling of the heart.

Human neural stem cell-derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway.

Human neural stem cell-derived extracellular vesicles exhibit analogous functions to their parental cells, and can thus be used as substitutes for stem cells in stem cell therapy, thereby mitigating the risks of stem cell therapy and advancing the frontiers of stem cell-derived treatments. This lays a foundation for the development of potentially potent new treatment modalities for ischemic stroke. However, the precise mechanisms underlying the efficacy and safety of human neural stem cell-derived extracellular vesicles remain unclear, presenting challenges for clinical translation. To promote the translation of therapy based on human neural stem cell-derived extracellular vesicles from the bench to the bedside, we conducted a comprehensive preclinical study to evaluate the efficacy and safety of human neural stem cell-derived extracellular vesicles in the treatment of ischemic stroke. We found that administration of human neural stem cell-derived extracellular vesicles to an ischemic stroke rat model reduced the volume of cerebral infarction and promoted functional recovery by alleviating neuronal apoptosis. The human neural stem cell-derived extracellular vesicles reduced neuronal apoptosis by enhancing phosphorylation of phosphoinositide 3-kinase, mammalian target of rapamycin, and protein kinase B, and these effects were reversed by treatment with a phosphoinositide 3-kinase inhibitor. These findings suggest that human neural stem cell-derived extracellular vesicles play a neuroprotective role in ischemic stroke through activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Finally, we showed that human neural stem cell-derived extracellular vesicles have a good in vivo safety profile. Therefore, human neural stem cell-derived extracellular vesicles are a promising potential agent for the treatment of ischemic stroke.

Light-gated integrator for highlighting kinase activity in living cells

Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

AoRan1 Is Involved in Regulating Conidiation, Stress Resistance, Secondary Metabolism, and Pathogenicity in Arthrobotrys oligospora

Arthrobotrys oligospora is a representative nematode-trapping (NT) fungus that is able to capture, kill, and digest nematodes by producing specialized three-dimensional networks (traps) under nutrient-deprived conditions. Ran1 is a serine/threonine protein kinase that can act as a negative regulator of sexual conjugation and meiosis. However, the specific role of Ran1 remains largely unknown in NT fungi. Here, we identified AoRan1 (AOL_s00004g277) via gene disruption, phenotypic analysis, and metabolomic analysis. Our findings reveal that Aoran1 knockout caused a remarkable increase in conidial production, traps, and nematode feeding efficiency. In addition, the absence of Aoran1 resulted in the accumulation of lipid droplets and increased autophagic levels as well as increased tolerance to cell wall synthesis-disturbing reagents and oxidants. Metabolomic analyses also suggested that AoRan1 is involved in multiple metabolic processes, such as fatty acid biosynthesis. In summary, our results suggest that AoRan1 is crucial in conidiation, pathogenicity, and secondary metabolism. This study’s results further our understanding of the molecular mechanisms by which AoRan1 regulates conidiation and trap formation in A. oligospora.

Insights into the mechanism of Danggui Liuhuang Tang in treating night sweats: A network pharmacology and molecular docking approach.

Night sweats can occur independently or in association with a number of medical conditions and can significantly disrupt daily life. This study focuses on the treatment of primary night sweats. Despite the considerable interest in Danggui Liuhuang Tang (DGLHT), an effective traditional Chinese medicine formula, its mechanism of action remains unknown. There is also no existing literature on the subject. Network pharmacology and molecular docking techniques. Network pharmacology techniques were employed to identify 109 active ingredients and 808 potential targets of DGLHT, as well as 2385 targets associated with night sweating diseases. The screening process yielded 375 common targets shared between DGLHT and night sweating. These included the active ingredients baicalein, quercetin, huarangiin, and tetrahydroafrican antipyrine, and the core targets interleukin 6, serine/threonine protein kinase 1, tumor necrosis factor, GAPDH enzyme, and Src protein kinase were identified. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DGLHT exerts its therapeutic effects primarily by modulating the PI3K-Akt signaling pathway, neuroactive ligand-receptor interactions, lipid metabolism, and atherosclerosis pathways. Molecular docking revealed strong binding activity between the main active ingredients and their potential targets. The research identifies promising active ingredients and targets related to the effectiveness of DGLHT in controlling night sweats, thus contributing to the further exploration of potential therapeutics for this condition. In addition, the results of this experiment provide a basis for future research into night sweats.

Empowering AlphaFold2 for protein conformation selective drug discovery with AlphaFold2-RAVE.

Small-molecule drug design hinges on obtaining co-crystallized ligand-protein structures. Despite AlphaFold2's strides in protein native structure prediction, its focus on apo structures overlooks ligands and associated holo structures. Moreover, designing selective drugs often benefits from the targeting of diverse metastable conformations. Therefore, direct application of AlphaFold2 models in virtual screening and drug discovery remains tentative. Here, we demonstrate an AlphaFold2-based framework combined with all-atom enhanced sampling molecular dynamics and Induced Fit docking, named AF2RAVE-Glide, to conduct computational model-based small-molecule binding of metastable protein kinase conformations, initiated from protein sequences. We demonstrate the AF2RAVE-Glide workflow on three different mammalian protein kinases and their type I and II inhibitors, with special emphasis on binding of known type II kinase inhibitors which target the metastable classical DFG-out state. These states are not easy to sample from AlphaFold2. Here, we demonstrate how with AF2RAVE these metastable conformations can be sampled for different kinases with high enough accuracy to enable subsequent docking of known type II kinase inhibitors with more than 50% success rates across docking calculations. We believe the protocol should be deployable for other kinases and more proteins generally.