Mitogen-activated Protein Kinase Family Research Articles

Mitogen-activated protein kinase kinase 7 in inflammatory, cancer, and neurological diseases.

Stress-activated mitogen-activated protein kinase kinase 7 (MKK7) is a member of the dual-specificity mitogen-activated protein kinase family. In the human body, MKK7 controls essential physiological processes, including but not limited to proliferation and differentiation in multiple tissues and organs. MKK7, along with the MKK4 pathway, has been implicated in stress-activated activities and biological events that are mediated by c-Jun N-terminal kinase (JNK) signaling. Although numerous studies have been performed to identify the role of JNK in multiple biological processes, there are limited publications that focus on dissecting the independent role of MKK7. Recent research findings have spurred testing via in vivo genetically deficient models, uncovering previously undocumented JNK-independent functions of MKK7. Here we discuss both JNK-dependent and-independent functions of MKK7 in vivo. This review summarizes the role of MKK7 in inflammation, cytokine production, cancer, and neurological diseases.

Genome wide identification and characterization of MAPK genes reveals their potential in enhancing drought and salt stress tolerance in Gossypium hirsutum

BackgroundThe cotton crop is universally considered as protein and edible oil source besides the major contributor of natural fiber and is grown in tropical and subtropical regions around the world Unpredicted environmental stresses are becoming significant threats to sustainable cotton production, ultimately leading to a substantial irreversible economic loss. Mitogen-activated protein kinase (MAPK) is generally considered essential for recognizing environmental stresses through phosphorylating downstream signal pathways and plays a vital role in numerous biological processes.ResultsWe have identified 74 MAPK genes across cotton, 41 from G. hirsutum, 19 from G. raimondii, whereas 14 have been identified from G. arboreum. The MAPK gene-proteins have been further studied to determine their physicochemical characteristics and other essential features. In this perspective, characterization, phylogenetic relationship, chromosomal mapping, gene motif, cis-regulatory element, and subcellular localization were carried out. Based on phylogenetic analysis, the MAPK family in cotton is usually categorized as A, B, C, D, and E clade. According to the results of the phylogenic relationship, cotton has more MAPKS genes in Clade A than Clade B. The cis-elements identified were classified into five groups (hormone responsiveness, light responsiveness, stress responsiveness, cellular development, and binding site). The prevalence of such elements across the promoter region of these genes signifies their role in the growth and development of plants. Seven GHMAPK genes (GH_A07G1527, GH_D02G1138, GH_D03G0121, GH_D03G1517, GH_D05G1003, GH_D11G0040, and GH_D12G2528) were selected, and specific tissue expression and profiling were performed across drought and salt stresses. Results expressed that six genes were upregulated under drought treatment except for GH_D11G0040 which is downregulated. Whereas all the seven genes have been upregulated at various hours of salt stress treatment.ConclusionsRNA sequence and qPCR results showed that genes as differentially expressed across both vegetative and reproductive plant parts. Similarly, the qPCR analysis showed that six genes had been upregulated substantially through drought treatment while all the seven genes were upregulated across salt treatments. The results of this study showed that cotton GHMPK3 genes play an important role in improving cotton resistance to drought and salt stresses. MAPKs are thought to play a significant regulatory function in plants' responses to abiotic stresses according to various studies. MAPKs' involvement in abiotic stress signaling and innovation is a key goal for crop species research, especially in crop breeding.

Fragment-Based and Structural Investigation for Discovery of JNK3 Inhibitors.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are related to cell proliferation, gene expression, and cell death. JNK isoform 3 (JNK3) is an important therapeutic target in varieties of pathological conditions including cancers and neuronal death. There is no approved drug targeting JNKs. To discover chemical inhibitors of JNK3, virtual fragment screening, the saturation transfer difference (STD) NMR, in vitro kinase assay, and X-ray crystallography were employed. A total of 27 fragments from the virtually selected 494 compounds were identified as initial hits via STD NMR and some compounds showed the inhibition of the activity of JNK3 in vitro. The structures of JNK3 with a fragment and a potent inhibitor were determined by X-ray crystallography. The fragment and inhibitor shared a common JNK3-binding feature. The result shows that fragment screening by NMR spectroscopy is a very efficient method to screen JNK3 binders and the structure of JNK3-inhibitor complex can be used to design and develop more potent inhibitors.

Upregulation of dual-specificity phosphatase-26 is required for transforming growth factor β1(TGFβ1)-induced Epithelial-mesenchymal transition in A549 and PANC1 cells.

Transforming Growth Factor β (TGFβ) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFβ-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFβ-induced EMT is largely unkonwn. Real-time qPCR analyses were performed to determine the effect of TGFβ1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFβ1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFβ1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFβ1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFβ1 on E-cadherin and mesenchymal genes in the cells. Data provided suggest that TGFβ1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFβ1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFβ1-associated EMT in tumor cells.

Role of the JNK Pathway in Bladder Cancer.

Bladder cancer, one of the most frequently diagnosed cancers worldwide, is associated with high morbidity and mortality and a poor prognosis. The bladder cancer types include 1) non-muscle invasive bladder cancer (NMIBC) and 2) muscle invasive bladder cancer (MIBC). Metastases and chemoresistance in MIBC patients are the leading causes of the high death rate. c-Jun N-terminal kinase (JNK) is an important factor for the undifferentiated state of cancer cells. JNK belongs to the mitogen-activated protein kinases (MAPKs) family; it is activated by various extracellular stimuli, such as stress, radiation, and growth factors and mediates diverse cellular functions, such as apoptosis, autophagy, proliferation, invasion, and migration by mediating AKT (Ak strain transforming), ATG (Autophagy related), mTOR (Mammalian target of rapamycin), and caspases 3, 8, and 9. This review describes the JNK-related functions, mechanisms, and signaling in bladder cancer.

Downregulated miRNAs associated with auditory deafferentation and compensatory neural plastic changes following single-sided deafness in the inferior colliculi of rats

Background and aimsDeafferentation and compensatory neural plastic changes in the inferior colliculus (IC) have been suggested following single-sided deafness (SSD). We explored related miRNA changes in the IC of SSD rats using miRNA microarray analyses. MethodsEight-week-old rats were divided into control and SSD rats (n = 8 for each group). SSD rats underwent right-side cochlear ablation surgery, with the IC harvested two weeks post-surgery. miRNA microarray analysis was performed using GeneChip miRNA 4.0, microarray (Affymetrix Inc.). miRNAs whose expression levels differed between SSD and control rats with a fold-change ≥ 1.5 and P < 0.05 were examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Target genes of differentially expressed miRNAs were predicted using TargetScan software. The pathways related to predicted target genes were analyzed. mRNA levels of predicted target genes were estimated using qRT-PCR. ResultsThe expression of miR-15b-5p, miR-202-5p, and miR-212-3p was lower in the contralateral (left) IC of SSD rats than that of control rats. In SSD rats, miRNA expression levels in the contralateral IC were 0.45-, 0.25-, and 0.50-fold lower for miR-15b-5p, miR-202-5p, and miR-212-3p, respectively (P < 0.05). The expression of predicted target genes (Spred1, Rasa1, Lsm11, and Srsf1) was higher in the contralateral IC of SSD rats than in control rats. The targets were predicted to be related with cleavage of growing transcripts in the termination region, mitogen-activated protein kinase family signaling cascades, RAF/AMP kinase cascade, regulation of RAS by GTPase activating proteins (GAPs), and RNA polymerase II transcription termination. For ipsilateral ICs, miR-425-3p, miR-199a-5p, and miR-134-3p showed lower expressions in SSD rats than in control rats, which were 0.55-, 0.61-, and 0.69-fold lower, respectively (P < 0.05). The expression of predicted target genes (Atp2b2, Grin2b, Foxp1, Ztbt20, Zfp91, and Strn) was higher in the ipsilateral IC of SSD rats; the regulation of synaptic plasticity, cAMP signaling pathway, metal ion binding, and calcium ion transport can be associated with these target genes. ConclusionAdult rats with unilateral auditory deprivation showed miRNA changes in the IC. The contralateral IC showed decreased miRNA expression predicted to be related to MAPK and RAS signaling, whereas the ipsilateral IC revealed decreased miRNA expression predicted to be associated with synaptic plasticity and calcium ion transport.

Salidroside inhibited cerebral ischemia/reperfusion-induced oxidative stress and apoptosis via Nrf2/Trx1 signaling pathway.

Cerebral ischemia reperfusion injury (CIRI) is still a serious problem threatening human health. Salidroside (SAL) is a natural phenylpropanoid glycoside compound with antioxidant, anti-inflammatory, and anti-ischemic properties. This study investigated the protective mechanism of SAL on middle cerebral artery occlusion (MCAO)- and oxygen-glucose deprivation/reoxygenation (OGD/R) model-induced CIRI via regulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/thioredoxin 1 (Trx1) axis. The results indicated that SAL (50mg/kg or 100mg/kg, intraperitoneal injection) not only effectively alleviated infarction rate, improved histopathological changes, relieved apoptosis by strengthening the suppression of cleaved caspase-3 and Bax/Bcl-2 proteins and decreased malondialdehyde (MDA) formation, but also increased superoxide dismutase (SOD) and catalase (CAT) activities and upregulated the expressions of Nrf2 and Trx1 on MCAO-induced CIRI rats. SAL also efficiently inhibited apoptosis and decreased oxidative stress in OGD/R-stimulated PC12 cells. Furthermore, blocking the Nrf2/Trx1 pathway using tretinoin, an Nrf2 inhibitor, significantly reversed the protective effect of SAL on OGD/R-induced oxidative stress. Moreover, SAL reduced the expression of apoptosis signal-regulating kinase-1 (ASK1) and mitogen-activated protein kinase (MAPK) family proteins. These results demonstrated that SAL inhibited oxidative stress through Nrf2/Trx1 signaling pathway, and subsequently reduced CIRI-induced apoptosis by inhibiting ASK1/MAPK.

New Therapeutic Approaches to and Mechanisms of Ginsenoside Rg1 against Neurological Diseases.

Neurological diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), stroke, cerebral infarction, ischemia-reperfusion injury, depression and, stress, have high incidence and morbidity and often lead to disability. However, there is no particularly effective medication against them. Therefore, finding drugs with a suitable efficacy, low toxicity and manageable effects to improve the quality of life of patients is an urgent problem. Ginsenoside Rg1 (Rg1) is the main active component of ginseng and has a variety of pharmacological effects. In this review, we focused on the therapeutic potential of Rg1 for improving neurological diseases. We introduce the mechanisms of Ginsenoside Rg1 in neurological diseases, including apoptosis, neuroinflammation, the microRNA (miRNA) family, the mitogen-activated protein kinase (MAPK) family, oxidative stress, nuclear factor-κB (NF-κB), and learning and memory of Rg1 in neurological diseases. In addition, Rg1 can also improve neurological diseases through the interaction of different signal pathways. The purpose of this review is to explore more in-depth ideas for the clinical treatment of neurological diseases (including PD, AD, HD, stroke, cerebral infarction, ischemia–reperfusion injury, depression, and stress). Therefore, Rg1 is expected to become a new therapeutic method for the clinical treatment of neurological diseases.

MdMKK9-Mediated the Regulation of Anthocyanin Synthesis in Red-Fleshed Apple in Response to Different Nitrogen Signals.

The mitogen-activated protein kinase (MAPK) signaling cascade is a widely existing signal transduction system in eukaryotes, and plays an important role in the signal transduction processes of plant cells in response to environmental stress. In this study, we screened MdMKK9, a gene in the MAPK family. This gene is directly related to changes in anthocyanin synthesis in the ‘Daihong’ variety of red-fleshed apple (Malus sieversii f neidzwetzkyana (Dieck) Langenf). MdMKK9 expression was up-regulated in ‘Daihong’ tissue culture seedlings cultured at low levels of nitrogen. This change in gene expression up-regulated the expression of genes related to anthocyanin synthesis and nitrogen transport, thus promoting anthocyanin synthesis and causing the tissue culture seedlings to appear red in color. To elucidate the function of MdMKK9, we used the CRISPR/Cas9 system to construct a gene editing vector for MdMKK9 and successfully introduced it into the calli of the ‘Orin’ apple. The MdMKK9 deletion mutants (MUT) calli could not respond to the low level of nitrogen signal, the expression level of anthocyanin synthesis-related genes was down-regulated, and the anthocyanin content was lower than that of the wild type (WT). In contrast, the MdMKK9-overexpressed calli up-regulated the expression level of anthocyanin synthesis-related genes and increased anthocyanin content, and appeared red in conditions of low level of nitrogen or nitrogen deficiency. These results show that MdMKK9 plays a role in the adaptation of red-fleshed apple to low levels of nitrogen by regulating the nitrogen status and anthocyanin accumulation.

TaMPK2B, a member of the MAPK family in T. aestivum, enhances plant low-Pi stress tolerance through modulating physiological processes associated with phosphorus starvation defensiveness

The mitogen-activated protein kinase (MAPK) cascades are present in plant species and modulate plant growth and stress responses. This study characterizes TaMPK2B, a MAPK family gene in T. aestivum that regulates plant adaptation to low-Pi stress. TaMPK2B harbors the conserved domains involving protein phosphorylation and protein-protein interaction. A yeast two-hybrid assay reveals an interaction between TaMPK2B and TaMPKK2 and between the latter and TaMPKKK;A, suggesting that all comprise a MAPK signaling cascade TaMPKKK;A-TaMPKK2-TaMPK2B. TaMPK2B expression levels were elevated in roots and leaves under a Pi starvation (PS) condition. Additionally, the induced TaMPK2B transcripts under PS in tissues were gradually restored following the Pi normal recovery condition. TaMPK2B overexpression conferred on plants improved PS adaptation; the tobacco lines with TaMPK2B overexpression enhanced the plant’s dry mass production, Pi uptake capacity, root system architecture (RSA) establishment, and ROS homeostasis relative to wild type under PS treatment. Moreover, the transcripts of genes in phosphate transporter (PT), PIN-FORMED, and antioxidant enzyme (AE) families, including NtPT3 and NtPT4, NtPIN9, and NtMnSOD1 and NtPOD1;7, were elevated in Pi-deprived lines overexpressing TaMPK2B. Transgene analyses validated their functions in regulating Pi uptake, RSA establishment, and AE activities of plants treated by PS. These results suggest that TaMPK2B-mediated plant PS adaptation is correlated with the modified transcription of distinct PT, PIN, and AE genes. Our investigation suggests that TaMPK2B is one of the crucial regulators in plant low-Pi adaptation by improving Pi uptake, RSA formation, and ROS homeostasis via transcriptionally regulating genes associated with the above physiological processes.

Genome-Wide Identification of MAPK, MAPKK, and MAPKKK Gene Families in Fagopyrum tataricum and Analysis of Their Expression Patterns Under Abiotic Stress

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signal transduction pathway, ubiquitous in eukaryotes, such as animals and plants. The MAPK cascade has a dominant role in regulating plant adaptation to the environment, such as through stress responses, osmotic adjustment, and processes that modulate pathogenicity. In the present study, the MAPK cascade gene family was identified in Fagopyrum tataricum (Tartary buckwheat), based on complete genome sequence data. Using phylogenetic tree, conservative motif, and chromosome location analyses, a total of 65 FtMAPK cascade genes, distributed on five chromosomes, were classified into three families: MAPK (n = 8), MAPKK (n = 1), and MAPKKK (n = 56). Transcriptome data from Tartary buckwheat seedlings grown under different light conditions demonstrated that, under blue and red light, the expression levels of 18 and 36 FtMAPK cascade genes were up-regulated and down-regulated, respectively. Through qRT-PCR experiments, it was observed that FtMAPK5, FtMAPKK1, FtMAPKKK8, FtMAPKKK10, and FtMAPKKK24 gene expression levels in the Tartary buckwheat seedlings increased under three types of abiotic stress: drought, salt, and high temperature. A co-expression network of FtMAPK cascade genes was constructed, based on gene expression levels under different light conditions, and co-expressed genes annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, which identified numerous transcription factors related to plant abiotic stress. The authors conclude that FtMAPK cascade genes have important roles in the growth and development of Tartary buckwheat, as well as its responses to abiotic stress.

Abstract 1804: IND-ready clinical candidate for HPK1 developed with excellent efficacy and safety profile

Abstract Background Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase (MAP4K) family of protein serine/threonine kinases1,2 and is a negative regulator of T and B cell receptor signaling3. Inhibition of HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment of solid tumors3. We present in vitro, in vivo, pharmacokinetic (PK) and early safety profiles of a novel and differentiated HPK1 inhibitor GRC 54276. Methods GRC 54276 is our clinical candidate, designed and developed using intuitive medicinal chemistry design and supported by computational approaches. SAR studies included a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo efficacy was demonstrated in syngeneic mouse tumor models, both as a single agent and combination with immune check-point blockers (ICB), mouse anti-CTLA4 antibody or Atezolizumab (human anti-PD-L1 antibody). ADME-PK properties was evaluated cross-species. GLP and non-GLP safety tolerability studies were conducted in mice and monkeys. Results GRC 54276 demonstrated sub-nanomolar HPK1 potency, strong target engagement of pSLP76 inhibition, anti-tumor cytokines (IL-2 and IFN-γ) induction, reversal of immunosuppression by prostaglandin E2 (PGE2) or adenosine in both human and mouse systems. GRC 54276 demonstrated very strong tumor growth inhibition (TGI) efficacy as single agent and significantly enhanced TGI in combination with ICB antibodies anti-CTLA4 (CT26 model) or Atezolizumab (MC38-hPD-L1 model). The in vivo TGI efficacy mechanistically correlated with increased immune responses of cytokine induction, infiltration of cytotoxic T cells, tumor rejections accompanied by immune memory T cells induction. Pharmacokinetic profile of GRC 54276 included cross-species oral bioavailability (30 to 100%), predominant clearance by CYP3A4 with no significant inhibition of major CYP isoforms, negative activation potency in human PXR assay at several-fold over EDmax exposures. Safety profile demonstrated that GRC 54276 is non-genotoxic in the bone marrow micronucleus assay in mice and has no hERG liability. The no observed adverse effect levels in the 14-day and 17-day exploratory studies in mice and monkeys were 50 and 15 mg/kg/day, respectively. Conclusions GRC 54276, our clinical candidate is potent, selective, orally bioavailable HPK1 inhibitor demonstrating strong single-agent and combination efficacy, low DDI liability accompanied by acceptable early safety profile in mice and monkeys. GRC 54276 is undergoing IND enabling studies to advance to Phase 1 clinical trial. Acknowledgements We thank Vidya Kattige, Pooja Sawant, Shital More, Rahul B. Bhadane, Ajit Jagadale, Sanjay Gaikwad, Pramod Sagar for their contributions to the project

TOPK Activation Exerts Protective Effects on Cisplatin-induced Acute Kidney Injury

ObjectiveT-LAK-cell-originated protein kinase (TOPK), a PSD95-Disc large-ZO1 (PDZ) binding kinase (PBK), is a novel member of the mitogen-activated protein kinase (MAPK) family. Studies have shown that TOPK plays a critical role in the function of tumor cells, including apoptosis and mitosis. However, little is known on the effect of TOPK in cisplatin-induced acute kidney injury (CP-AKI). This study aimed to investigate the role and mechanism of TOPK in CP-AKI.MethodsCisplatin was administered to C57BL/6 mice and cultured kidney tubular epithelial cells (TECs) to establish the CP-AKI murine or cellular models. TECs were then stimulated with the specific inhibitor of TOPK OTS514 or transfected with the recombinant-activated plasmid TOPK-T9E to inhibit or activate TOPK. The TECs were treated with AKT inhibitor VIII following stimulation with OTS514 or cisplatin. Western blotting and flow cytometry were used to evaluate the cell cycle and apoptosis of TECs.ResultsThe analysis revealed that the TOPK activity was significantly suppressed by cisplatin, both in vivo and in vitro. Furthermore, the pharmacological inhibition of TOPK by OTS514, a specific inhibitor of TOPK, exacerbated the cisplatin-induced cell cycle arrest in the G2/M phase and apoptosis of cultured TECs. Moreover, the TOPK activation via the TOPK-T9E plasmid transfection could partially reverse the cell cycle arrest at the G2/M phase and apoptosis of cisplatin-treated TECs. In addition, AKT/protein kinase B (PKB), as a TOPK target protein, was inhibited by cisplatin in cultured TECs. The pharmaceutical inhibition of AKT further aggravated the apoptosis of TECs induced by cisplatin or TOPK inhibition. TOPK systematically mediated the apoptosis via the AKT pathway in the CP-AKI cell model.ConclusionThese results indicate that TOPK activation protects against CP-AKI by ameliorating the G2/M cell cycle arrest and cell apoptosis.

The analgesic effects of β-elemene in rats with neuropathic pain by inhibition of spinal astrocytic ERK activation.

Neuropathic pain takes a heavy toll on individual well-being, while current therapy is far from desirable. Herein, we assessed the analgesic effect of β-elemene, a chief component in the traditional Chinese medicine Curcuma wenyujin, and explored the underlying mechanisms at the level of spinal dorsal horn (SDH) under neuropathic pain. A spared nerve injury (SNI)-induced neuropathic pain model was established in rats. Intraperitoneal injection (i.p.) of β-elemene was administered for 21 consecutive days. Mechanical allodynia was explored by von Frey filaments. The activation of the mitogen-activated protein kinase (MAPK) family (including ERK, p38, and JNK) in spinal neurons, astrocytes, and microglia was evaluated using immunostaining 29 days after SNI surgery. The expression of GFAP, Iba-1, p-ERK, p-JNK, and p-p38 within the SDH was measured using immunoblotting. The levels of proinflammatory cytokines (including TNF-α, IL-1β, and IL-6) were measured with ELISA. The levels of oxidative stress indicators (including MDA, SOD, and GSH-PX) were detected using biochemical tests. Consecutive i.p. administration of β-elemene relieved SNI-induced mechanical allodynia (with an EC50 of 16.40 mg/kg). SNI significantly increased the expression of p-ERK in spinal astrocytes but not microglia on day 29. β-elemene reversed spinal astrocytic ERK activation and subsequent upregulation of proinflammatory cytokines in SNI rats, with no effect on the expression of p38 and JNK in spinal glia. β-elemene also exerted antioxidative effects by increasing the levels of SOD and GSH-PX and decreasing the level of MDA. Our results suggest that SNI induces robust astrocytic ERK activation within the SDH in the late phase of neuropathic pain. β-elemene exerts remarkable analgesic effects on neuropathic pain, possibly by inhibiting spinal astrocytic ERK activation and subsequent neuroinflammatory processes. Our findings suggest that β-elemene might be a promising analgesic for the treatment of chronic pain.

Inhibitory potential of phytochemicals from Chromolaena odorata L. against apoptosis signal-regulatory kinase 1: A computational model against colorectal cancer

Apoptosis signal kinase 1 (ASK 1) is a member of the mitogen-activated protein kinase (MAPK) family that induces cells apoptosis including colorectal cancer (CRC). CRC is the second most common type of malignancy globally. Hence, ASK 1 plays an essential role in the pathogenesis of CRC and therefore, is an exclusive target in drug design and discovery for CRC. Herein, applied computational approaches including molecular docking, molecular mechanics/generalized born surface area calculation and pharmacokinetic models were performed to propose putative ASK 1 antagonists from natural compounds. Seven (7) ligands were identified as potent inhibitors and two top hit compounds were validated using molecular dynamics (MD) simulation studies. The density function theory (DFT) of the hits were performed at the Becke three Lee Yang Parr/6-31G(d) level of theory to understand their molecular reactivity. Seven compounds identified as ASK 1 antagonists have docking score ranging from −9.10 to −8.14 kcal/mol which is comparable to the reference ligand camptosar (-7.03 kcal/mol). One of the compounds, odoratin has finally emerged as the structurally stable compound with −9.10 kcal/mol and MD simulation over 50 ns indicated that odoratin forms stable interaction with key amino acid residues such as LEU 686, VAL 757 and PRO 758. DFT study showed that the studied compounds have proton donating and accepting ability hence, potent inhibitory and solubility effects. The findings from this study suggest that, odoratin could be considered a potent ASK 1 inhibitor and could be experimentally verified as a lead compound for search of MAPK inhibitors for colorectal cancer.

Agomelatine rescues lipopolysaccharide-induced neural injury and depression-like behaviors via suppression of the Gαi-2-PKA-ASK1 signaling pathway

BackgroundAgomelatine has been shown to be effective in the treatment of depression, but the molecular mechanisms underlying its antidepressant effects have yet to be elucidated. Identification of these molecular mechanisms would not only offer new insights into the basis for depression but also provide the foundation for the development of novel treatments for this disorder.MethodsIntraperitoneal injection of LPS was used to induce depression-like behaviors in rats. The interactions of the 5-HT2C reporter and Gαi-2 were verified by immunoprecipitation or immunofluorescence assay. Inflammatory related proteins, autophagy related proteins and apoptosis markers were verified by immunoblotting or immunofluorescence assay. Finally, electron microscopy analysis was used to observe the synapse and ultrastructural pathology.ResultsHere, we found that the capacity for agomelatine to ameliorate depression and anxiety in a lipopolysaccharide (LPS)-induced rat model of depression was associated with an alleviation of neuroinflammation, abnormal autophagy and neuronal apoptosis as well as the promotion of neurogenesis in the hippocampal dentate gyrus (DG) region of these rats. We also found that the 5-HT2C receptor is coupled with G alphai (2) (Gαi-2) protein within hippocampal neurons and, agomelatine, acting as a 5-HT2C receptor antagonist, can up-regulate activity of the Gαi-2-cAMP-PKA pathway. Such events then suppress activation of the apoptosis signal-regulating kinase 1 (ASK1) pathway, a member of the mitogen-activated protein kinase (MAPK) family involved in pathological processes of many diseases.ConclusionTaken together, these results suggest that agomelatine plays a neuroprotective role in regulating neuroinflammation, autophagy disorder and apoptosis in this LPS-induced rat model of depression, effects which are associated with the display of antidepressant behaviors. These findings provide evidence for some of the potential mechanisms for the antidepressant effects of agomelatine.Graphical

Progress on the role of mixed lineage kinase 3 in cardiovascular diseases

<p indent="0mm">Despite great changes in the spectrum of human diseases, cardiovascular diseases (CVDs) have been the leading cause of human death worldwide for more than <sc>20 years.</sc> According to a report from the World Health Organization, an estimated 17.9 million people die of CVDs every year, accounting for 32% of total deaths worldwide. Developing countries are those with the greatest loss of healthy life years. China is the largest developing country in the world, and deaths from CVDs in China have ranked first among total causes of death for urban and rural residents for many years. According to the latest <italic>Annual Report on Cardiovascular Health and Diseases in China (2021)</italic>, the number of patients with CVDs in China has reached 330 million, which has brought a heavy disease burden to the national economy and medical system. Therefore, further exploration of the potential pathogenesis and possible therapeutic targets of CVDs is of great significance for disease prevention and human health promotion. Mixed lineage kinase 3 (MLK3, also known as MAP3K11) is a member of the mitogen-activated protein kinase (MAPK) family. It is an important molecule in the cell life cycle and regulates many critical pathophysiological processes such as cell proliferation, differentiation, apoptosis, and energy metabolism through c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. In addition, it is involved in the initiation and development processes of numerous types of tumors in the digestive system, nervous system, reproductive system, and some other physiological systems. It is noteworthy that recent studies suggest that MLK3 is also closely associated with CVDs, and plays a significant role in the pathophysiological processes of CVDs such as heart failure, myocardial hypoxic injury, and sepsis-induced cardiomyopathy. It has been found that MLK3 is likely to be involved in the initiation and development of these diseases through activation of the inflammatory response, damage from reactive oxygen species, induction of cell apoptosis, and through its influence on metabolic and energy homeostasis. However, the role of MLK3 in different CVDs differs; in recent studies, some of its effects are controversial and need to be further verified. Overall, MLK3 is likely to be a potential therapeutic target for CVDs. Here, we provide a general overview and summarize related signaling pathways of MLK3 in the physiological state, while elucidating the mechanisms of MLK3 in common CVDs based on the latest research results. This provides a baseline for broadening research and exploring preventive and therapeutic ideas in CVDs.

Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain cysteine residues that are predicted to be oxidized by H2O2 that lie within or in close proximity to the substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following treatment with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pretreatment, both ERK2 and p38α exhibited an increase in the relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcatfor both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). Likewise, fluorescence polarization assays suggest that oxidation of ERK2 increases its affinity for some ligands while decreasing its affinity for others. Interestingly, the upstream MAPK kinase, MEK1, also recognizes ERK1/2 via interactions with the DRS, suggesting that ERK1/2 activation dynamics may be affected by signal‐generated H2O2 within the cellular environment. Indeed, cell‐based assays suggested that pre‐treatment of NIH‐3T3 cells with the cell permeable H2O2 scavenger, PEG‐catalase, alters ERK1/2 phosphorylation following platelet‐derived growth factor (PDGF) treatment. Together, these findings suggest that redox modification of the cysteines located within the MAPK DRS alters the activation dynamics and may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.

Association between MAP3K4 gene polymorphisms and the risk of schizophrenia susceptibility in a Northeast Chinese Han population.

Schizophrenia stands out as one of the most devastating psychiatric disorders. Previous findings have shown that schizophrenia is a polygenic genetic disorder. Thus, abnormal neurodevelopment and neurogenesis may be associated with the etiology of schizophrenia, so genes which affect these processes may be potential candidate genes of schizophrenia. Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene is a member of the mitogen-activated protein kinase family. Taking into account previous findings, MAP3K4 plays a crucial role in the fundamental pathology of various nervous system diseases. In the present study, we aim to explore the association of MAP3K4 and schizophrenia in an independent case-control sample including 627 schizophrenic patients and 1175 healthy controls from a Northeast Chinese Han population. Both the allelic and genotypic association analyses showed that 6 SNPs in MAP3K4 were significantly associated with schizophrenia (rs590988, rs625977, rs9295134, rs12110787, rs1001808 and rs9355870). After rigorous Bonferroni correction, 4 SNPs (rs9295134, rs12110787, rs1001808 and rs9355870) were still significantly associated with the disease. The haplotype composed of these four SNPs also showed significantly global and individual association with schizophrenia. These results suggest that MAP3K4 is a susceptibility gene for schizophrenia in the Northeast Chinese Han population.

Colocalization and Interaction Study of Neuronal JNK3, JIP1, and β-Arrestin2 Together with PSD95.

c-Jun N-terminal kinases (JNKs) are stress-activated serine/threonine protein kinases belonging to the mitogen-activated protein kinase (MAPK) family. Among them, JNK3 is selectively expressed in the central nervous system, cardiac smooth muscle, and testis. In addition, it is the most responsive JNK isoform to stress stimuli in the brain, and it is involved in synaptic dysfunction, an essential step in neurodegenerative processes. JNK3 pathway is organized in a cascade of amplification in which signal transduction occurs by stepwise, highly controlled phosphorylation. Since different MAPKs share common upstream activators, pathway specificity is guaranteed by scaffold proteins such as JIP1 and β-arrestin2. To better elucidate the physiological mechanisms regulating JNK3 in neurons, and how these interactions may be involved in synaptic (dys)function, we used (i) super-resolution microscopy to demonstrate the colocalization among JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2 in cultured hippocampal neurons, and (ii) co-immunoprecipitation techniques to show that the two scaffold proteins and JNK3 can be found interacting together with PSD95. The protein-protein interactions that govern the formation of these two complexes, JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2, may be used as targets to interfere with their downstream synaptic events.