Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition

Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition.     

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2β, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.     

Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae

The vacuoles of the yeast Saccharomyces cerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa B signaling pathway.     

The Puccinia striiformis effector Hasp98 facilitates pathogenicity by blocking the kinase activity of wheat TaMAPK4

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) employs virulence effectors to disturb host immunity and causes devastating stripe rust disease. However, our understanding of how Pst effectors regulate host defense responses remains limited. In this study, we determined that the Pst effector Hasp98, which is highly expressed in Pst haustoria, inhibits plant immune responses triggered by flg22 or nonpathogenic bacteria. Overexpression of Hasp98 in wheat (Triticum aestivum) suppressed avirulent Pst-triggered immunity, leading to decreased H₂O₂ accumulation and promoting P. striiformis infection, whereas stable silencing of Hasp98 impaired P. striiformis pathogenicity. Hasp98 interacts with the wheat mitogen-activated protein kinase TaMAPK4, a positive regulator of plant resistance to stripe rust. The conserved TEY motif of TaMAPK4 is important for its kinase activity, which is required for the resistance function. We demonstrate that Hasp98 inhibits the kinase activity of TaMAPK4 and that the stable silencing of TaMAPK4 compromises wheat resistance against P. striiformis. These results suggest that Hasp98 acts as a virulence effector to interfere with the MAPK signaling pathway in wheat, thereby promoting P. striiformis infection.     

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.     

MiR-942-3p as a Potential Prognostic Marker of Gastric Cancer Associated with AR and MAPK/ERK Signaling Pathway

Gastric cancer is a common tumor with high morbidity and mortality. MicroRNA (miRNA) can regulate gene expression at the translation level and various tumorigenesis processes, playing an important role in tumor occurrence and prognosis. This study aims to screen miRNA associated with gastric cancer prognosis as biomarkers and explore the regulatory genes and related signaling pathways. In this work, R language was used for the standardization and differential analysis of miRNA and mRNA expression profiles. Samples were randomly divided into a testing group and a training group. Subsequently, we built the five miRNAs (has-miR-9-3p, has-miR-135b-3p, has-miR-143-5p, has-miR-942-3p, has-miR-196-3p) prognostic modules, verified and evaluated their prediction ability by the Cox regression analysis. They can be used as an independent factor in the prognosis of gastric cancer. By predicting and analyzing potential biological functions of the miRNA target genes, this study found that the AR gene was not only a hub gene in the PPI network, but also associated with excessive survival of patients. In conclusion, this study demonstrated that hsa-miR-942-3p could be a potential prognostic marker of gastric cancer associated with the AR and MAPK/ERK signaling pathways. The results of this study provide insights into the occurrence and development of gastric cancer.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

Copper-induced germline apoptosis in Caenorhabditis elegans: The independent roles of DNA damage response signaling and the dependent roles of MAPK cascades

Excess copper is toxic to life. Copper has been shown to induce apoptosis in various cell lines and tissues. However, due to the lack of appropriate gene knockout animal models, data concerning the underlying pathways of copper-induced apoptosis are insufficient, especially with regards to in vivo systems. The nematode Caenorhabditis elegans is a good model to study basic biological processes, including stress responses and apoptosis. In the present study, we investigated copper-induced germline apoptosis in the C. elegans strains carrying mutated alleles of homologs to known mammalian genes that are involved in apoptosis regulation. We show here that exposing C. elegans to copper causes dose- and time-dependent germline apoptosis. The knockout of checkpoint genes hus-1, clk-2, the Bcl-2 homolog ced-9, and the BH3-only domain egl-1 did not prevent cells of the germline from copper-induced apoptosis. The loss-of-function of the tumor suppressor gene, p53/cep-1, caused a significant increase in germline apoptosis with exposure to copper, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. The knockout of the caspase gene ced-3 and the Apaf-1 homolog ced-4 abrogated both copper-induced and physiological germline apoptosis. Germline apoptosis stopped increase in the strains lin-45(ku51), mek-2(n1989), mpk-1(ku1) under copper stresses, respectively. Copper-induced apoptosis was blocked in the loss-of-function alleles of both JNK and p38 MAPK cascades excepting pmk-3, one of the three p38 MAPK components. Together, the results of this study suggest that caspase and Apaf-1 are required for copper-induced germline apoptosis while DNA damage response genes are not essential, and that the Raf-MEK-ERK, ASK1/2-MKK7-JNK, ASK1/2-MKK3/6-p38 signaling pathways are indispensable in mediating this apoptotic response.