Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

ML241 Antagonizes ERK 1/2 Activation and Inhibits Rotavirus Proliferation.

Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.

ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

OBJECTIVE: This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. MATERIALS AND METHODS: Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. RESULTS: BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. CONCLUSION: This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity.

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Circ-LDLRAD3/miR-655-3p/MAPK1 axis enhances cell migration and invasion in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a prevalent histological subtype of thyroid cancer, whose occurrence and development may be related to circRNA dysregulation. This research proposed to unravel circ-LDLRAD3-related mechanisms in PTC. First, circ-LDLRAD3, miR-655-3p .and MAPK1 levels in PTC were quantitatively measured. Then, plasmid vectors or oligonucleotides that interfere with circ-LDLRAD3, miR-655-3p, or MAPK1 were transfected into PTC cells, followed by the analysis of proliferation, apoptosis, migration, and invasion. Finally, the targeted binding sites between miR-655-3p and circ-LDLRAD3 or MAPK1 were predicted by starBase and experimentally verified. Statistically, PTC samples expressed high circ-LDLRAD3 and MAPK1 and low miR-655-3p. Knocking down circ-LDLRAD3 or enhancing miR-655-3p hindered PTC cell proliferation, migration, and invasion, and forced apoptosis. circ-LDLRAD3 bound to miR-655-3p to affect MAPK1 expression. Elevating MAPK1 rescued circ-LDLRAD3 knockdown-allowed obstruction of PTC cell growth. In conclusion, circ-LDLRAD3 stimulates PTC development by releasing miR-655-3p-targeted MAPK1.

Transient Receptor Potential Ankyrin 1-dependent Activation of Extracellular Signal-regulated Kinase 2 in the Cerebral Cortices Contributes to Cortical Spreading Depolarization.

Extracellular signal-regulated kinase (ERK) are serine/threonine-selective proteins and ERK1/2 can be phosphorylated in peripheral and central brain regions after cortical spreading depolarization (CSD) and calcitonin gene-related peptide; However, it remains unclear about whether and how ERK activity modulates CSD that correlates to migraine aura. Here, we determined the role of ERK in regulating CSD and explored the underlying mechanism involving transient receptor potential ankyrin 1 (TRPA1), a stress-sensing cation channel. CSD was recorded using intrinsic optical imaging in mouse brain slices, and electrophysiology in rats. Phosphorylated ERK (pERK1/2) and interleukin-1ß (IL-1ß) protein levels were detected using Western blot or enzyme-linked immunosorbent assay, respectively. IL-1ß mRNA level was detected using qPCR. The results showed that an ERK inhibitor, SCH77298, markedly prolonged CSD latency and reduced propagation rate in mouse brain slices. Corresponding to this, CSD induction increased levels of cytosolic pERK1/2 in ipsilateral cerebral cortices of rats, the elevation of which correlated to the level of IL-1ß mRNA. Mechanistic analysis showed that pre-treatment of an anti-TRPA1 antibody reduced the cytosolic pERK2 level but not pERK1 following CSD in cerebral cortices of rats and this level of pERK2 correlated with that of cerebral cortical IL-1ß protein. Furthermore, an ERK activator, AES16-2M, but not its scrambled control, reversed the prolonged CSD latency by a TRPA1 inhibitor, HC-030031, in mouse brain slices. These data revealed a crucial role of ERK activity in regulating CSD, and elevation of pERK and IL-1ß production induced by CSD is predominantly TRPA1 channel-dependent, thereby contributing to migraine pathogenesis.

MAPK1 Mediates MAM Disruption and Mitochondrial Dysfunction in Diabetic Kidney Disease via the PACS-2-Dependent Mechanism.

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD). Mitochondrial dysfunction in renal tubules, occurring early in the disease, is linked to the development of DKD, although the underlying pathways remain unclear. Here, we examine diabetic human and mouse kidneys, and HK-2 cells exposed to high glucose, to show that high glucose disrupts mitochondria-associated endoplasmic reticulum membrane (MAM) and causes mitochondrial fragmentation. We find that high glucose conditions increase mitogen-activated protein kinase 1(MAPK1), a member of the MAP kinase signal transduction pathway, which in turn lowers the level of phosphofurin acidic cluster sorting protein 2 (PACS-2), a key component of MAM that tethers mitochondria to the ER. MAPK1-induced disruption of MAM leads to mitochondrial fragmentation but this can be rescued in HK-2 cells by increasing PACS-2 levels. Functional studies in diabetic mice show that inhibition of MAPK1 increases PACS-2 and protects against the loss of MAM and the mitochondrial fragmentation. Taken together, these results identify the MAPK1-PACS-2 axis as a key pathway to therapeutically target as well as provide new insights into the pathogenesis of DKD.

N6-methyladenosine-modified CircPSMA7 enhances bladder cancer malignancy through the miR-128-3p/MAPK1 axis.

Several studies have indicated that circular RNAs (circRNAs) play vital roles in the progression of various diseases, including bladder cancer (BCa). However, the underlying mechanisms by which circRNAs drive BCa malignancy remain unclear. In this study, we identified a novel circRNA, circPSMA7 (circbaseID:has_circ_0003456), showing increased expression in BCa cell lines and tissues, by integrating the reported information with circRNA-seq and qRT-PCR. We revealed that circPSMA7 is associated with a higher tumor grade and stage in BCa. M6A modification was identified in circPSMA7, and IGF2BP3 recognized this modification and stabilized circPSMA7, subsequently increasing the circPSMA7 expression. In vitro and in vivo experiments showed that circPSMA7 promoted BCa proliferation and metastasis by regulating the cell cycle and EMT processes. CircPSMA7 acted as a sponge for miR-128-3p, which showed antitumor effects in BCa cell lines, increasing the expression of MAPK1. The tumor proliferation and metastasis suppression induced by silencing circPSMA7 could be partly reversed by miR-128-3p inhibition. Thus, the METTL3/IGF2BP3/circPSMA7/miR-128-3p/MAPK1 axis plays a critical role in BCa progression. Furthermore, circPSMA7 may be a potential diagnostic biomarker and novel therapeutic target for patients with BCa.

MEK1-ERK1/2 signaling regulates the cardiomyocyte non-sarcomeric actin cytoskeletal network.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic ß- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic ß-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Expression and promoter methylation of mitogen-activated protein kinase 1 in tumor and marginal cells of breast cancer.

AIM: In the present study, we sought to explore potential differences in the expression and promoter methylation of mitogen-activated protein kinase 1 (MAPK1) between tumor and marginal cells of breast cancer lesions. METHODS: A total of 50 randomly selected patients with breast cancer (BCa) undergoing needle biopsy were enrolled. Clinical specimens containing both tumor and marginal cells were collected and preserved. After DNA extraction using specific primers, MAPK1 mRNA and promoter methylation were measured with spectrophotometry at 260/280 nm absorption wavelengths. To deliver a comparative analysis, data from The Cancer Genome Atlas (TCGA) program regarding breast cancer (BRCA), were downloaded from Xena Functional Genomics Explorer and separately analyzed. The suitability of MAPK1 expression and promoter methylation as biomarkers for BCa was analyzed with receiver operating characteristic (ROC) curves. RESULTS: We found a positive correlation between tumor stage and MAPK1 expression (P-value: 0.029) in BCa. Likewise, MAPK1 expression was significantly associated with lymph node metastasis (P-value: 0.018). There was a significant difference in the expression of MAPK1 mRNA between tumor and marginal cells of BCa and BRCA (P-value < 0.001). However, we did not find any statistically significant difference in MAPK1 promoter methylation between tumor and marginal cells of both BCa and BRCA. With an area under the curve (AUC) of 0.71, the diagnostic accuracy of MAPK1 expression in BCa and BRCA was validated. However, MAPK1 promoter methylation was not found to be a suitable biomarker. CONCLUSION: Our findings suggest that while MAPK1 expression, might be a promising biomarker for evaluating oncogenic activity in patients suspected of BCa. We were not able to detect a prognostic/diagnostic role for MAPK1 promoter methylation.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Integrated bioinformatics and network pharmacology to explore the therapeutic target and molecular mechanisms of Taxus chinensis against non-small cell lung cancer.

Taxus chinensis (TC) has tremendous therapeutic potential in alleviating non-small cell lung cancer (NSCLC), but the mechanism of action of TC remains unclear. Integrated bioinformatics and network pharmacology were employed in this study to explore the potential targets and molecular mechanism of TC against NSCLC. Data obtained from public databases were combined with appropriate bioinformatics tools to identify the common targets for TC and NSCLC. Common targets were uploaded to the Metascape database for gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway analyses. A protein-protein interaction network was established, and topological analysis was performed to obtain hub genes. The expression of the hub genes in NSCLC tissues and their consequent effects on the prognosis of patients with NSCLC were confirmed using the Human Protein Atlas database and appropriate bioinformatics tools. Molecular docking was used to verify the binding affinity between the active ingredients and hub targets. We found 401 common targets that were significantly enriched in the cancer, MAPK signaling, and PI3K/Akt signaling pathways. Proto-oncogene tyrosine-protein kinase Src (SRC), mitogen-activated protein kinase 1, phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1), AKT serine/threonine kinase 1 (AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and lymphocyte-specific protein tyrosine kinase were identified as the hub genes. Immunohistochemical results confirmed that the expression of SRC, mitogen-activated protein kinase 1, PIK3R1, AKT1, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha was upregulated in the NSCLC tissues, while survival analysis revealed the expression of SRC, AKT1, PIK3R1, and lymphocyte-specific protein tyrosine kinase was closely related to the prognosis of patients with NSCLC. Molecular docking results confirmed all bioactive ingredients present in TC strongly bound to hub targets. We concluded that TC exhibits an anti-NSCLC role through multi-target combination and multi-pathway cooperation.

Activation Loop Plasticity and Active Site Coupling in the MAP Kinase, ERK2.

Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) µs, totaling 727 µs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 µs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.

Oryza sativa L. Indica Seed Coat Ameliorated Concanavalin A-Induced Acute Hepatitis in Mice via MDM2/p53 and PKCα/MAPK1 Signaling Pathways.

Acute hepatitis (AH) is a common liver disease with an increasing number of patients each year, requiring the development of new treatments. Hence, our work aimed to evaluate the therapeutic effect of Oryza sativa L. indica (purple rice) seed coat on concanavalin A (ConA)-induced AH and further reveal its potential mechanisms. Purple rice seed coat extract (PRE) was extracted with hydrochloric acid ethanol and analyzed through a widely targeted components method. We evaluated the effects of PRE on AH through histopathological examination, liver function, gut microbiota composition, and the intestinal barrier. The potential targets of PRE on AH were predicted by bioinformatics. Western blotting, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining, and corresponding kits were used to investigate PRE effects on predicting targets and associated signaling pathways in AH mice. In AH model mice, PRE treatment increased transformed mouse 3T3 cell double minute 2 (MDM2) expression to inhibit apoptosis; it also markedly downregulated protein kinase C alpha (PKCα), prostaglandin-endoperoxide synthase 1 (PTGS1), and mitogen-activated protein kinase 1 (MAPK1) activity to alleviate inflammation. Thus, PRE treatment also recovered the intestinal barrier, decreased the lipopolysaccharide (LPS) levels of plasma and the liver, enhanced liver function, and improved the composition of intestinal microbiota. In general, PRE targeting MDM2, PKCα, MAPK1, and PTGS1 ameliorated ConA-induced AH by attenuating inflammation and apoptosis, restoring the intestinal barrier, enhancing the liver function, and improving the gut microbiota, which revealed that the purple rice seed coat might hold possibilities as a therapeutic option for AH.

The complex impact of cancer-related missense mutations on the stability and on the biophysical and biochemical properties of MAPK1 and MAPK3 somatic variants.

Mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3), also called extracellular regulated kinases (ERK2 and ERK1), are serine/threonine kinase activated downstream by the Ras/Raf/MEK/ERK signal transduction cascade that regulates a variety of cellular processes. A dysregulation of MAPK cascade is frequently associated to missense mutations on its protein components and may be related to many pathologies, including cancer. In this study we selected from COSMIC database a set of MAPK1 and MAPK3 somatic variants found in cancer tissues carrying missense mutations distributed all over the MAPK1 and MAPK3 sequences. The proteins were expressed as pure recombinant proteins, and their biochemical and biophysical properties have been studied in comparison with the wild type. The missense mutations lead to changes in the tertiary arrangements of all the variants. The thermodynamic stability of the wild type and variants has been investigated in the non-phosphorylated and in the phosphorylated form. Significant differences in the thermal stabilities of most of the variants have been observed, as well as changes in the catalytic efficiencies. The energetics of the catalytic reaction is affected for all the variants for both the MAPK proteins. The stability changes and the variation in the enzyme catalysis observed for most of MAPK1/3 variants suggest that a local change in a residue, distant from the catalytic site, may have long-distance effects that reflect globally on enzyme stability and functions.

MAPK1 promotes the metastasis and invasion of gastric cancer as a bidirectional transcription factor.

BACKGROUND: The Mitogen-activated protein kinase 1 (MAPK1) has both independent functions of phosphorylating histones as a kinase and directly binding the promoter regions of genes to regulate gene expression as a transcription factor. Previous studies have identified elevated expression of MAPK1 in human gastric cancer, which is associated with its role as a kinase, facilitating the migration and invasion of gastric cancer cells. However, how MAPK1 binds to its target genes as a transcription factor and whether it modulates related gene expressions in gastric cancer remains unclear. RESULTS: Here, we integrated biochemical assays (protein interactions and chromatin immunoprecipitation (ChIP)), cellular analysis assays (cell proliferation and migration), RNA sequencing, ChIP sequencing, and clinical analysis to investigate the potential genomic recognition patterns of MAPK1 in a human gastric adenocarcinoma cell-line (AGS) and to uncover its regulatory effect on gastric cancer progression. We confirmed that MAPK1 promotes AGS cells invasion and migration by regulating the target genes in different directions, up-regulating seven target genes (KRT13, KRT6A, KRT81, MYH15, STARD4, SYTL4, and TMEM267) and down-regulating one gene (FGG). Among them, five genes (FGG, MYH15, STARD4, SYTL4, and TMEM267) were first associated with cancer procession, while the other three (KRT81, KRT6A, and KRT13) have previously been confirmed to be related to cancer metastasis and migration. CONCLUSION: Our data showed that MAPK1 can bind to the promoter regions of these target genes to control their transcription as a bidirectional transcription factor, promoting AGS cell motility and invasion. Our research has expanded the understanding of the regulatory roles of MAPK1, enriched our knowledge of transcription factors, and provided novel candidates for cancer therapeutics.