Mitogen-activated Protein Kinase Mutant Research Articles

Chemical Knockdown of Phosphorylated p38 Mitogen-Activated Protein Kinase (MAPK) as a Novel Approach for the Treatment of Alzheimer's Disease.

Targeted protein degradation (TPD) provides unique advantages over gene knockdown in that it can induce selective degradation of disease-associated proteins attributed to pathological mutations or aberrant post-translational modifications (PTMs). Herein, we report a protein degrader, PRZ-18002, that selectively binds to an active form of p38 MAPK. PRZ-18002 induces degradation of phosphorylated p38 MAPK (p-p38) and a phosphomimetic mutant of p38 MAPK in a proteasome-dependent manner. Given that the activation of p38 MAPK plays pivotal roles in the pathophysiology of Alzheimer's disease (AD), selective degradation of p-p38 may provide an attractive therapeutic option for the treatment of AD. In the 5xFAD transgenic mice model of AD, intranasal treatment of PRZ-18002 reduces p-p38 levels and alleviates microglia activation and amyloid beta (Aβ) deposition, leading to subsequent improvement of spatial learning and memory. Collectively, our findings suggest that PRZ-18002 ameliorates AD pathophysiology via selective degradation of p-p38, highlighting a novel therapeutic TPD modality that targets a specific PTM to induce selective degradation of neurodegenerative disease-associated protein.

Abstract 3365: Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone

Abstract Overactivation of mitogen-activated protein kinase (MAPK) signaling is a frequently observed driver of many cancers. Targeted MAPK pathway inhibitors have been approved as therapies in patient populations with genomic alterations to MAPK pathway genes (e.g. dabrafenib in BRAF-mutant melanoma), with multiple other MAPK-targeted therapies in development. We have previously demonstrated that cell lines with MAPK pathway genomic alterations show increased sensitivity to ASTX029, our potent and selective dual-mechanism ERK inhibitor which is currently undergoing clinical development. However, MAPK oncogene status alone is not a universal indicator of sensitivity, nor is it clear that genomic markers of sensitivity to single agent inhibition represent the optimal approach to predicting response to MAPK targeted agents in the combination setting (e.g. sotorasib + trametinib). We therefore analysed MAPK pathway related transcriptional signatures to assess predictability for response to ASTX029 in combination with a SHP2 inhibitor. We previously presented data from large-scale viability screens assessing the response to ASTX029 as a single agent treatment (437 cell lines), as well as in combination with an inhibitor of SHP2 (491 cell lines), an upstream adaptor protein in the MAPK pathway. Using these data, several published transcriptional signatures related to the MAPK pathway were assessed for their ability to stratify sensitive vs resistant cell lines when benchmarked against MAPK oncogene genomic status alone. All assessed transcriptomics-based scores had a significant positive correlation with growth inhibition for ERK inhibitor single agent treatment as well as in combination with a SHP2 inhibitor. Transcriptomics-based scores were shown to be more sensitive than the use of mutant MAPK oncogene status alone based on assessment of binomial regression models. The significant increase in sensitivity enables the identification of additional cell lines responsive to MAPK pathway inhibition. The sensitivity was further increased when considering response to the combination of an ERK and SHP2 inhibitor compared to the ERK inhibitor as a single agent. This analysis demonstrates the potential use of MAPK pathway-related transcriptomics-based scores for the stratification of MAPK pathway inhibitor response. ASTX029 is currently undergoing clinical development in advanced solid tumours (NCT03520075) and transcriptomics-based methods may provide utility in refining patient selection in the clinic for single agent or combination approaches. Citation Format: Jessica Brothwood, Andrea Biondo, Kim-Hien Dao, Keisha Hearn, Chris Hindley, Navrohit Kandola, Harold Keer, John Lyons, Yoko Nakatsuru, Marc O'Reilly, Nicola Wallis, Harpreet Saini. Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3365.

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.

Direct targeting of the downstream MAPK pathway to suppress ERK activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including EGFR-inhibition. To elucidate the interplay between EGF signaling and ERK activation in tumors, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumors, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved FRET-based ERK biosensor EKAREN5. We reveal that oncogene-driven signaling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance.

The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.IMPORTANCEAspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkCA. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

Differential roles, crosstalk and response to the Antifungal Protein AfpB in the three Mitogen-Activated Protein Kinases (MAPK) pathways of the citrus postharvest pathogen Penicillium digitatum

Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.

Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance.

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiae slt2Δ, bck1Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants (sakAΔ, mpkCΔ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion

Mitogen-activated protein kinases (MAPKs) play a central role in transferring signals and regulating gene expression in response to extracellular stimuli. An ortholog of the Saccharomyces cerevisiae cell wall integrity MAPK was identified in the phytopathogenic fungus Sclerotinia sclerotiorum. Disruption of the S. sclerotiorum Smk3 gene severely reduced virulence on intact host plant leaves but not on leaves stripped of cuticle wax. This was attributed to alterations in hyphal apical dominance leading to the inability to aggregate and form infection cushions. The mutation also caused loss of the ability to produce sclerotia, increased aerial hyphae formation, and altered hyphal hydrophobicity and cell wall integrity. Mutants had slower radial expansion rates on solid media but more tolerance to elevated temperatures. Loss of the SMK3 cell wall integrity MAPK appears to have impaired the ability of S. sclerotiorum to sense its surrounding environment, leading to misregulation of a variety of functions. Many of the phenotypes were similar to those observed in S. sclerotiorum adenylate cyclase and SMK1 MAPK mutants, suggesting that these signaling pathways co-regulate aspects of fungal growth, physiology, and pathogenicity.

The Caenorhabditis elegans p38 MAPK Gene plays a key role in protection from mycobacteria.

Mitogen‐activated protein kinases (MAPK) are critical mediators of cellular responses to pathogens and are activated in response to infection, but investigation is difficult in multi‐cell hosts due to developmental lethality of mutations. Mycobacterium marinum (Mm) is an established model for tuberculosis, a disease afflicting nearly one‐third of the world's population. We found that Mm‐infected Caenorhabditis elegans display >80% mortality, but nonpathogenic M. smegmatis cause <15% mortality. C. elegans display pathological changes when infected with Mm, whereas Mm mutants produce lower mortality, suggesting that C. elegans is a promising virulence model for detailed genetic analysis. C. elegans MAPK mutants are hypersusceptible to mycobacterial infection; however, the C. elegans TOL‐like, TGF‐β and insulin‐like pathway genes do not play important roles in susceptibility. We show that pathogenic mycobacteria inhibit MAPK‐mediated protection through the MAPK phosphatase gene and demonstrate that C. elegans provide a genetically tractable pathogenicity model of both the host and pathogen.

Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity.

Disruption of cell wall integrity system should be an effective strategy for control of fungal pathogens. To augment the cell wall disruption efficacy of monoterpenoid phenols (carvacrol, thymol), antimycotic potency of benzaldehyde derivatives that can serve as chemosensitizing agents were evaluated against strains of Saccharomyces cerevisiae wild type (WT), slt2Δ and bck1Δ (mutants of the mitogen-activated protein kinase (MAPK) and MAPK kinase kinase, respectively, in the cell wall integrity pathway). Among fourteen compounds investigated, slt2Δ and bck1Δ showed higher susceptibility to nine benzaldehydes, compared to WT. Differential antimycotic activity of screened compounds indicated “structure-activity relationship” for targeting the cell wall integrity, where 2-hydroxy-4-methoxybenzaldehyde (2H4M) exhibited the highest antimycotic potency. The efficacy of 2H4M as an effective chemosensitizer to monoterpenoid phenols (viz., 2H4M + carvacrol or thymol) was assessed in yeasts or filamentous fungi (Aspergillus, Penicillium) according to European Committee on Antimicrobial Susceptibility Testing or Clinical Laboratory Standards Institute M38-A protocols, respectively. Synergistic chemosensitization greatly lowers minimum inhibitory or fungicidal concentrations of the co-administered compounds. 2H4M also overcame the tolerance of two MAPK mutants (sakAΔ, mpkCΔ) of Aspergillus fumigatus to fludioxonil (phenylpyrrole fungicide). Collectively, 2H4M possesses chemosensitizing capability to magnify the efficacy of monoterpenoid phenols, which improves target-based (viz., cell wall disruption) antifungal intervention.

Hairy cell leukemia cell lines lack signature BRAF mutations of typical disease and MAP2K1 mutations of atypical disease

Tumor-derived cell lines provide important models of disease but require validation with regard to parental disease. Hairy cell leukemia (HCL) is a rare B cell leukemia with characteristic hair-like cytoplasmic projections on tumor cells and they uniquely express multiple surface immunoglobulin (sIg) isotypes. A mutation [V(600)E] in exon 15 of BRAF, a signal transduction protein kinase, has recently been identified as near universal in typical or classic HCL (HCLc), but absent in atypical variant HCL (HCLv) and HCLc expressing the rearranged IGHV4-34 gene. In a few HCLc cases lacking the [V(600)E] mutation, alternative mutations in [D(449)E] and [F(468)C] have been reported in BRAF. We previously examined 5 HCL cell lines used as relevant disease models, Hair-M, HCLL-7876, EH, Eskol, HC-1, and HCLv-07 (the latter of known HCLv origin) and found that while they recapitulated phenotypic and functional characteristics of HCLc, they lacked the definitive BRAF [V(600)E] mutation, as did HCLv-07. More recently, whole exome next-generation sequencing studies in HCLv and HCLc/IGHV4-34 disease subsets have identified recurrent mutations in the MAP2K1 gene in ∼50% of atypical cases, all lacking the BRAF [V(600)E] mutation. MAP2K1 encodes MEK1, a dual-specificity kinase that is a direct effector of BRAF. A mutant mitogen-activated protein kinase (MAPK) pathway emerges as an important driver of neoplastic transformation in HCLc and in HCLv and HCLc/IGHV4-34 disease. We examined the reported MAP2K1 mutations and alternative BRAF mutations in each of the HCL cells lines. In 5/5 lines, no MAP2K1 or alternative BRAF mutations were found, including in the HCLv cell line. These data indicate derivation of known HCL cell lines from atypical HCL disease that lack MAP2K1 mutations, and suggest caution in their experimental use to represent specific HCL subsets. They further substantiate the importance of whole genome sequencing in providing robust genetic markers to validate disease models, in addition to identifying driver mutations relevant to understanding disease origins.

In Vivo Interaction Proteomics Reveal a Novel p38 Mitogen-Activated Protein Kinase/Rack1 Pathway Regulating Proteostasis in Drosophila Muscle

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.

Identification of constitutively active AtMPK6 mutants using a functional screen in Saccharomyces cerevisiae.

MAPK (Mitogen-Activated Protein Kinases) mutants which are active independently of phosphorylation by upstream MAPK Kinases (MAPKKs) help to clarify signal transduction processes through MAPK modules and provide a useful tool to understand MAPK roles in the cell. The identification of such mutations is tricky. In this chapter, we provide a detailed protocol for their screening, taking advantage of a functional expression assay in yeast.

Endothelin-1 enhances cell migration through COX-2 up-regulation in human chondrosarcoma

BackgroundChondrosarcoma is a type of highly malignant tumor with a potent capacity of local invasion and distant metastasis. The effect of endothelin-1 (ET-1) on migration activity in human chondrosarcoma cells is not clearly understood. Here, we found that ET-1 increased the migration and expression of cyclooxygenase (COX)-2 in human chondrosarcoma cells. MethodsET-1-mediated COX-2 expression was assessed by qPCR and Western blot analysis. The mechanisms of action of ET-1 in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the COX-2 promoter. ResultsHuman chondrosarcoma tissues had significant expression levels of ET-1 and COX-2, which were higher than that in normal cartilage. Exogenous ET-1 increased cell migration and the expression of COX-2. In addition, COX-2 protein levels and cell migration ability were abolished by ET receptor antagonists. Activation of the mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways after ET-1 treatment was demonstrated, and ET-1-induced COX-2 expression and cell migration activity were inhibited by the specific inhibitor and mutant of MAPK and AP-1 cascades. ET-1 increased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Furthermore, knockdown of ET-1 decreased cell metastasis in vitro and in vivo. ConclusionsOur results indicated that ET-1 enhances the cell migration of chondrosarcoma by increasing COX-2 expression through the ET receptors, MAPK, and AP-1 signal transduction pathway. General significanceWe link high ET-1 and COX-2 expression to chondrosarcoma.

Two guard cell‐preferential MAPKs, MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)-induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca(2+) ]cyt ) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL-induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL-induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca(2+) ]cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca(2+) ]cyt oscillation in YEL-induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.

Role and contribution of Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Immunity during Proteus vulgaris infection

The p38 mitogen‐activated protein kinase (MAPK) pathway is highly conserved signaling cascade that converts into various outputs. C. elegans innate immunity also requires a conserved MAPK pathway that regulates the basal and pathogen‐induced expression of immune effectors. As an opportunistic pathogen, Proteus vulgaris does not cause death in wild type C. elegans. To understand the role and contribution of MAPK pathway, two MAPK mutant C. elegans were infected with P. vulgaris. The infections were performed on solid (full‐lawn) and liquid assays with wild type and MAPK mutant C. elegans (AU37 and KU25). P. vulgaris required 66±5h for AU37 and 55±5h for KU25 mutants for the complete killing in liquid assays. However, it required 10 days for the killing of both mutants in solid plate assays. To evaluate the role of innate immune pathways specific players at the mRNA level, the regulation of few candidate antimicrobial genes were kinetically investigated. Real‐time PCR analysis revealed the regulation of candidate immune regulatory genes namely, CUB‐like proteins (F08G5.6), lysozyme (lys‐7), CUB‐like proteins (F08G5.6), neuropeptide‐like factors (nlp‐29) and transcription factors of MAPK (ATF‐7) and daf‐2–daf‐16 (daf‐16) insulin‐like signaling pathways and MAPK pathway player during the course of P. vulgaris infections, indicating the role and contribution of MAPK pathway when compared to other innate immune pathways during the host response against the P. vulgaris infections.

Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

BackgroundDisruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy.MethodsBenzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI).ResultsSeveral benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective inhibition of fungal growth can also be achieved using combinations of these benzaldehydes.ConclusionsNatural benzaldehydes targeting cellular antioxidation components of fungi, such as superoxide dismutases, glutathione reductase, etc., effectively inhibit fungal growth. They possess antifungal or chemosensitizing capacity to enhance efficacy of conventional antifungal agents. Chemosensitization can reduce costs, abate resistance, and alleviate negative side effects associated with current antifungal treatments.

Sphingosine-1-phosphate에 의한 중간엽 줄기세포의 이동과 평활근세포로의 분화

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of Ca 2+ ([Ca 2+ ]i) and pretreatment with VPC23019, an antagonist of S1P₁/S1P₃, blocked S1P-induced migration and increase of [Ca 2+ ]i. Small interfering RNA-mediated knockdown of endogenous S1P1 attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced α-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and α-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an S1P1-p38 MAPK-dependent mechanism.

Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells

BackgroundLegionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells.ResultsWild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter.ConclusionsTaken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

Platelet-Activating Factor Receptor Mediates Oxidized Low Density Lipoprotein-Induced Migration of Bone Marrow-Derived Mesenchymal Stem Cells

Background: Oxidized low density lipoprotein (oxLDL) is involved in the development of vascular diseases. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyero-3-phosphorylcholine) is a key component of oxLDL. Methods: In the present study, we evaluate whether oxLDL can regulate migration of human bone-marrow derived stem cells (hBMSCs) and characterize the role of PAF in the oxLDL-induced migration. Results and Conclusions: oxLDL stimulated chemotaxis of hBMSCs in vitro. Treatment of the cells with BN52021, a specific antagonist of PAF receptor (PAF-R), completely blocked the cell migration induced by PAF, but not platelet-derived growth factor (PDGF-BB). Using PAF-R-specific small interfering RNA, it was demonstrated that silencing of endogenous PAF-R expression significantly attenuated cell migration induced by PAF, but not PDGF-BB, suggesting the specific involvement of PAF-R in the oxLDL-induced cell migration. In addition, PAF-induced migration of hBMSCs was abrogated by pretreating cells with mitogen-activated protein kinase (MAPK) inhibitors, including the MEK inhibitor U0126, the p38 MAPK inhibitor SB202190, and the JNK inhibitor SP600125. Moreover, adenoviral overexpression of a dominant negative mutant of p38 MAPK blocked PAF-stimulated migration. Taken together, these results suggest that PAF plays a pivotal role in the oxLDL-induced recruitment of hBMSCs through mechanisms involving PAF-R-dependent activation of MAPKs.

The role of mitogen‐activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea

In all fungi studied so far, mitogen-activated protein (MAP) kinase cascades serve as central signalling complexes that are involved in various aspects of growth, stress response and infection. In this work, putative components of the yeast Fus3/Kss1-type MAP kinase cascade and the putative downstream transcription factor Ste12 were analysed in the grey mould fungus Botrytis cinerea. Deletion mutants of the MAP triple kinase Ste11, the MAP kinase kinase Ste7 and the MAP kinase adaptor protein Ste50 all resulted in phenotypes similar to that of the previously described BMP1 MAP kinase mutant, namely defects in germination, delayed vegetative growth, reduced size of conidia, lack of sclerotia formation and loss of pathogenicity. Mutants lacking Ste12 showed normal germination, but delayed infection as a result of low penetration efficiency. Two differently spliced ste12 transcripts were detected, and both were able to complement the ste12 mutant, except for a defect in sclerotium formation, which was only corrected by the full-sized transcript. Overexpression of the smaller ste12 transcript resulted in delayed germination and strongly reduced infection. Bc-Gas2, a homologue of Magnaporthe grisea Gas2 that is required for appressorial function, was found to be non-essential for growth and infection, but its expression was under the control of both Bmp1 and Ste12. In summary, the role and regulatory connections of the Fus3/Kss1-type MAP kinase cascade in B. cinerea revealed both common and unique properties compared with those of other plant pathogenic fungi, and provide evidence for a regulatory link between the BMP1 MAP kinase cascade and Ste12.