Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway.

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate mitogen-activated protein kinase (MAPK) activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high-osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on a high-osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain. IMPORTANCE: Legionella pneumophila grows in a membrane-bound compartment in macrophages during disease. Construction of the compartment requires a dedicated secretion system that translocates virulence proteins into host cells. One of these proteins, LegA7, is shown to activate a stress response pathway in host cells called the mitogen-activated protein kinase (MAPK) pathway. The effects on the mammalian MAPK pathway were reconstructed in yeast, allowing the development of a strategy to identify the role of individual domains of LegA7. A domain similar to cysteine proteases is demonstrated to be critical for impinging on the MAPK pathway, and the catalytic activity of this domain is required for targeting this path. In addition, a conserved series of repeats, called ankyrin repeats, controls this activity. Data are provided that argue the interaction of the ankyrin repeats with unknown targets probably results in activation of the cysteine protease domain.

Repurposing approved protein kinase inhibitors as potent anti-leishmanials targeting Leishmania MAP kinases.

AIMS: Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors. MATERIALS AND METHODS: MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania, including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden. KEY FINDINGS: 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models. SIGNIFICANCE: Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance.

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

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

Modulation of mitochondrial function by extracellular acidosis in tumor cells and normal fibroblasts: Role of signaling pathways.

In many tumors pronounced extracellular acidosis resulting from glycolytic metabolism is found. Since several environmental stress factors affect the mitochondrial activity the aim of the study was to analyze the impact of acidosis on cellular oxygen consumption and which signaling pathways may be involved in the regulation. In two tumor cell lines and normal fibroblasts cellular oxygen consumption rate (OCR) and mitochondrial function were measured after 3 h at pH 6.6. Besides the activation of ERK1/2, p38 and PI3K signaling in the cytosolic and mitochondrial compartment, the mitochondrial structure and proteins related to mitochondria fission were analyzed. The acidic extracellular environment increased OCR in tumor cells but not in fibroblasts. In parallel, the mitochondrial membrane potential increased at low pH. In both tumor lines (but not in fibroblasts), the phosphorylation of ERK1/2 and PI3K/Akt was significantly increased, and both cascades were involved in OCR modulation. The activation of signaling pathways was located predominantly in the mitochondrial compartment of the cells. At low pH, the mitochondrial structure in tumor cells showed structural changes related to elongation whereas mitochondria fragmentation was reduced indicating mitochondria fusion. However, these morphological changes were not related to ERK1/2 or PI3K signaling. Acidic stress seems to induce an increased oxygen consumption, which might further aggravate tumor hypoxia. Low pH also induces mitochondria fusion that is not mediated by ERK1/2 or PI3K signaling. The mechanism by which these signaling cascades modulate the respiratory activity of tumor cells needs further investigation.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Interferon inducible guanylate-binding protein 1 modulates the lipopolysaccharide-induced cytokines/chemokines and mitogen-activated protein kinases in macrophages.

Guanylate-binding proteins (GBPs) are a family of interferon (IFN)-inducible GTPases and play a pivotal role in the host immune response to microbial infections. These are upregulated in immune cells after recognizing the lipopolysaccharides (LPS), the major membrane component of Gram-negative bacteria. In the present study, the expression pattern of GBP1-7 was initially mapped in phorbol 12-myristate 13-acetate-differentiated human monocytes THP-1 and mouse macrophages RAW 264.7 cell lines stimulated with LPS. A time-dependent significant expression of GBP1-7 was observed in these cells. Moreover, among the various GBPs, GBP1 has emerged as a central player in regulating innate immunity and inflammation. Therefore, to study the specific role of GBP1 in LPS-induced inflammation, knockdown of the Gbp1 gene was carried out in both cells using small interfering RNA interference. Altered levels of different cytokines (interleukin [IL]-4, IL-10, IL-12ß, IFN-γ, tumor necrosis factor-α), inducible nitric oxide synthase, histocompatibility 2, class II antigen A, protein kinase R, and chemokines (chemokine (C-X-C motif) ligand 9 [CXCL9], CXCL10, and CXCL11) in GBP1 knockdown cells were reported compared to control cells. Interestingly, the extracellular-signal-regulated kinase 1/2 mitogen-activated protein (MAP) kinases and signal transducer and activator of transcription 1 (STAT1) transcription factor levels were considerably induced in knockdown cells compared to the control cells. However, no change in the level of phosphorylated nuclear factor-kB, c-Jun, and p38 transcription factors was observed in GBP1 knockdown cells compared to the control cells. This study concludes that GBP1 may alter the expression of cytokines, chemokines, and effector molecules mediated by MAP kinases and STAT1 transcription factors.

Gochujang, a traditional Korean fermented food, protects through suppressed inflammatory pathways and histological structure disruption in dextran sodium sulfate (DSS)-induced colitis mice.

The mechanisms underlying chronic inflammatory diseases remain unclear. Therefore, researchers have explored the mechanisms underlying colitis using diverse materials. Recently, there has been an increasing interest in fermented products and bioconversion materials, their potential efficacy is being actively studied. Gochujang, a traditional Korean fermented product, is crafted by blending fermented Meju powder, gochu (Korean chili) powder, glutinous rice, and salt. In our study, we explored the effectiveness of Gochujang (500 mg/kg; Cheongju and Hongcheon, Korea) in dextran sulfate sodium (DSS)-induced colitis mice model. Gochujang was orally administered for 2 weeks, followed by the induction of colitis using 3% DSS in the previous week. During our investigation, Gochujang variants (TCG22-25, Cheongju and TCG22-48, Hongcheon) did not exhibit significant inhibition of weight reduction (p = 0.061) but notably (p = 0.001) suppressed the reduction in large intestine length in DSS-induced colitis mice. In the serum from colitis mice, TCG22-48 demonstrated reduced levels of the inflammatory cytokines interleukin (IL)-6 (p = 0.001) and tumor necrosis factor (TNF)-α (p = 0.001). Additionally, it inhibited the phosphorylation of Erk (p = 0.028), p38, and NF-κB (p = 0.001) the inflammatory mechanism. In our study, TCG22-25 demonstrated a reduction in the IL-6 level (p = 0.001) in serum and inhibited the phosphorylation of p38 and NF-κB (p = 0.001). Histological analysis revealed a significant (p = 0.001) reduction in the pathological score of the large intestine from TCG22-25 and TCG22-48. In conclusion, the intake of Gochujang demonstrates potent anti-inflammatory effects, mitigating colitis by preventing the large intestine length reduction of animals with colitis, lowering serum levels of TNF-α and IL-6 cytokines, and inhibiting histological disruption and inflammatory mechanism phosphorylation.

TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway.

OBJECTIVE: Src homology phosphotyrosin phosphatase 2 (SHP2) has been implicated in the progression of several cancer types. However, its function in endometrial cancer (EC) remains unclear. Here, we report that the ten-eleven translocation 3 (TET3)-mediated DNA demethylation modification is responsible for the oncogenic role of SHP2 in EC and explore the detailed mechanism. METHODS: The transcriptomic differences between EC tissues and control tissues were analyzed using bioinformatics tools, followed by protein-protein interaction network establishment. EC cells were treated with shRNA targeting SHP2 alone or in combination with isoprocurcumenol, an epidermal growth factor receptor (EGFR) signaling activator. The cell biological behavior was examined using cell counting kit-8, colony formation, flow cytometry, scratch assay, and transwell assays, and the median inhibition concentration values to medroxyprogesterone acetate/gefitinib were calculated. The binding of TET3 to the SHP2 promoter was verified. EC cells with TET3 knockdown and combined with SHP2 overexpression were selected to construct tumor xenografts in mice. RESULTS: TET3 and SHP2 were overexpressed in EC cells. TET3 bound to the SHP2 promoter, thereby increasing the DNA hydroxymethylation modification and activating SHP2 to induce the EGFR/extracellular signal-regulated kinase (ERK) pathway. Knockdown of TET3 or SHP2 inhibited EC cell malignant aggressiveness and impaired the EGFR/ERK pathway. Silencing of TET3 inhibited the tumorigenic capacity of EC cells, and ectopic expression of SHP2 or isoprocurcumenol reversed the inhibitory effect of TET3 knockdown on the biological activity of EC cells. CONCLUSION: TET3 promoted the DNA demethylation modification in the SHP2 promoter and activated SHP2, thus activating the EGFR/ERK pathway and leading to EC progression.

MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

TGFß1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFß1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase.

Following wounding, endogenously secreted TGFßs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFß effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFß. TGFß also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFß1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFß1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFß1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFß1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10-15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFß pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.

Insights from the molecular docking and simulation analysis of P38 MAPK phytochemical inhibitor complexes.

It is of interest to develop p38α MAPK inhibitors. Docking, ADMET properties calculation, molecular dynamics, and MM-PBSA approaches were used to investigate the therapeutic potentials of p38α MAPK in complex with SB203580 (1A9U). The photo-molecules metergoline, withaphysacarpin, philadelphicalactone, canthin-6-one 9-glucoside, and SB-21600011 demonstrated high binding affinity compared to the reference drug. Furthermore, ADME profiles validated the drug-like properties of the prioritized phyto-compounds. Besides that, MD simulations were performed along with reference inhibitors for withaphysacarpin and metergoline to assess stability. Binding free energy calculations (MM-PBSA) revealed that metergoline and withaphysacarpin had estimated values (G) of 97.151 ± 21.023 kJ/mol and -82.084 ± 15.766 kJ/mol, respectively. In this study, metergoline and withaphysacarpin were found to have high affinity against p38α MAPK when compared to the reference compound SB 203580.

MAP kinases may mediate regulation of the cell cycle in rice by E2F2 phosphorylation.

E2F is the key transcription factor that determines the proliferative status of cells by regulating the G1/S phase of the cell cycle. In this study, we show that in rice (Oryza sativa), OsE2F2 is a phosphorylation target of MAP kinases. The MAP kinases OsMPK3, OsMPK4, and OsMPK6 interact with and phosphorylate OsE2F2. Next, we determined the serine and threonine residues that could play a role in the phosphorylation of OsE2F2. Subsequently, our study suggests a possible link between MAP kinase-mediated OsE2F2 phosphorylation and its impact on DNA proliferation in the roots of rice seedlings. Finally, we found positive feedback regulation of OsMPK4 by OsE2F2. Therefore, our study hints at the potential impact of MAP kinase signaling on the cell cycle of rice plants.

From ribosome to ribotoxins: understanding the toxicity of deoxynivalenol and Shiga toxin, two food borne toxins.

Ribosomes that synthesize proteins are among the most central and evolutionarily conserved organelles. Given the key role of proteins in cellular functions, prokaryotic and eukaryotic pathogens have evolved potent toxins to inhibit ribosomal functions and weaken their host. Many of these ribotoxin-producing pathogens are associated with food. For example, food can be contaminated with bacterial pathogens that produce the ribotoxin Shiga toxin, but also with the fungal ribotoxin deoxynivalenol. Shiga toxin cleaves ribosomal RNA, while deoxynivalenol binds to and inhibits the peptidyl transferase center. Despite their distinct modes of action, both groups of ribotoxins hinder protein translation, but also trigger other comparable toxic effects, which depend or not on the activation of the ribotoxic stress response. Ribotoxic stress response-dependent effects include inflammation and apoptosis, whereas ribotoxic stress response-independent effects include endoplasmic reticulum stress, oxidative stress, and autophagy. For other effects, such as cell cycle arrest and cytoskeleton modulation, the involvement of the ribotoxic stress response is still controversial. Ribotoxins affect one organelle yet induce multiple toxic effects with multiple consequences for the cell. The ribosome can therefore be considered as the cellular "Achilles heel" targeted by food borne ribotoxins. Considering the high toxicity of ribotoxins, they pose a substantial health risk, as humans are highly susceptible to widespread exposure to these toxins through contaminated food sources.

Oleanolic Acid Complexation with Cyclodextrins Improves Its Cell Bio-Availability and Biological Activities for Cell Migration.

Wound healing is a complex process to restore skin. Plant-derived bioactive compounds might be a source of substances for the treatment of wounds stalled in a non-resolving stage of wound healing. Oleanolic acid (OA), a pentacyclic triterpene, has shown favorable wound healing properties both in vitro and in vivo. Unfortunately, OA cannot be solubilized in aqueous media, and it needs to be helped by the use of dimethyl sulfoxide (DMSO). In this paper, we have shown that cyclodextrins (CDs) are a good alternative to DMSO as agents to deliver OA to cells, providing better features than DMSO. Cyclodextrins are natural macromolecules that show a unique tridimensional structure that can encapsulate a wide variety of hydrophobic compounds. We have studied the cyclodextrin-encapsulated form of OA with OA/DMSO, comparing their stability, biological properties for cell migration, and cell viability. In addition, detailed parameters related to cell migration and cytoskeletal reorganization have been measured and compared. Our results show that OA-encapsulateds compound exhibit several advantages when compared to non-encapsulated OA in terms of chemical stability, migration enhancement, and preservation of cell viability.

Entanglement of MAPK pathways with gene expression and its omnipresence in the etiology for cancer and neurodegenerative disorders.

Mitogen Activated Protein Kinase (MAPK) is one of the most well characterized cellular signaling pathways that controls fundamental cellular processes including proliferation, differentiation, and apoptosis. These cellular functions are consequences of transcription of regulatory genes that are influenced and regulated by the MAP-Kinase signaling cascade. MAP kinase components such as Receptor Tyrosine Kinases (RTKs) sense external cues or ligands and transmit these signals via multiple protein complexes such as RAS-RAF, MEK, and ERKs and eventually modulate the transcription factors inside the nucleus to induce transcription and other regulatory functions. Aberrant activation, dysregulation of this signaling pathway, and genetic alterations in any of these components results in the developmental disorders, cancer, and neurodegenerative disorders. Over the years, the MAPK pathway has been a prime pharmacological target, to treat complex human disorders that are genetically linked such as cancer, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current review re-visits the mechanism of MAPK pathways in gene expression regulation. Further, a current update on the progress of the mechanistic understanding of MAPK components is discussed from a disease perspective.

Mitogen-Activated Protein Kinases (MAPKs) and Enteric Bacterial Pathogens: A Complex Interplay.

Diverse extracellular and intracellular cues activate mammalian mitogen-activated protein kinases (MAPKs). Canonically, the activation starts at cell surface receptors and continues via intracellular MAPK components, acting in the host cell nucleus as activators of transcriptional programs to regulate various cellular activities, including proinflammatory responses against bacterial pathogens. For instance, binding host pattern recognition receptors (PRRs) on the surface of intestinal epithelial cells to bacterial pathogen external components trigger the MAPK/NF-κB signaling cascade, eliciting cytokine production. This results in an innate immune response that can eliminate the bacterial pathogen. However, enteric bacterial pathogens evolved sophisticated mechanisms that interfere with such a response by delivering virulent proteins, termed effectors, and toxins into the host cells. These proteins act in numerous ways to inactivate or activate critical components of the MAPK signaling cascades and innate immunity. The consequence of such activities could lead to successful bacterial colonization, dissemination, and pathogenicity. This article will review enteric bacterial pathogens' strategies to modulate MAPKs and host responses. It will also discuss findings attempting to develop anti-microbial treatments by targeting MAPKs.

A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression.

Cancer cells often adapt to targeted therapies, yet the molecular mechanisms underlying adaptive resistance remain only partially understood. Here, we explore a mechanism of RAS/RAF/MEK/ERK (MAPK) pathway reactivation through the upregulation of RAF isoform (RAFs) abundance. Using computational modeling and in vitro experiments, we show that the upregulation of RAFs changes the concentration range of paradoxical pathway activation upon treatment with conformation-specific RAF inhibitors. Additionally, our data indicate that the signaling output upon loss or downregulation of one RAF isoform can be compensated by overexpression of other RAF isoforms. We furthermore demonstrate that, while single RAF inhibitors cannot efficiently inhibit ERK reactivation caused by RAF overexpression, a combination of two structurally distinct RAF inhibitors synergizes to robustly suppress pathway reactivation.

Anti-Fibrotic Effects of RF Electric Currents.

Hypertrophic scars and keloids are two different manifestations of excessive dermal fibrosis and are caused by an alteration in the normal wound-healing process. Treatment with radiofrequency (RF)-based therapies has proven to be useful in reducing hypertrophic scars. In this study, the effect of one of these radiofrequency therapies, Capacitive Resistive Electrical Transfer Therapy (CRET) on biomarkers of skin fibrosis was investigated. For this, in cultures of human myofibroblasts treated with CRET therapy or sham-treated, proliferation (XTT Assay), apoptosis (TUNEL Assay), and cell migration (Wound Closure Assay) were analyzed. Furthermore, in these cultures the expression and/or localization of extracellular matrix proteins such as α-SMA, Col I, Col III (immunofluorescence), metalloproteinases MMP1 and MMP9, MAP kinase ERK1/2, and the transcription factor NFκB were also investigated (immunoblot). The results have revealed that CRET decreases the expression of extracellular matrix proteins, modifies the expression of the metalloproteinase MMP9, and reduces the activation of NFκB with respect to controls, suggesting that this therapy could be useful for the treatment of fibrotic pathologies.

The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G2-Checkpoint Activation in Cells Exposed to Ionizing Radiation.

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.