When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)Hdependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

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Objective-NADH/NADPH oxidase is an important source of reactive oxygen species (ROS) in the vasculature. Recently, we demonstrated that p22 phox , an essential component of this oxidase, was expressed in human coronary arteries and that its expression was enhanced with the progression of atherosclerosis. The present study was undertaken to investigate its functional importance in the pathogenesis of coronary artery disease. For this aim, the expression of p22 phox , the distribution of oxidized low density lipoprotein (LDL), and the generation of ROS in directional coronary atherectomy (DCA) specimens were examined. Methods and Results-DCA specimens were obtained from patients with stable or unstable angina pectoris. The distribution of p22 phox and of oxidized LDL was examined by immunohistochemistry. The generation of superoxide in DCA specimens was assessed by the dihydroethidium method and lucigenin-enhanced chemiluminescence. ROS were closely associated with the distribution of p22 phox and oxidized LDL. Not only inflammatory cells but also smooth muscle cells and fibroblasts generated ROS. There was a correlation between ROS and the expression of p22 phox or oxidized LDL. The generation of ROS was significantly higher in unstable angina pectoris compared with stable angina pectoris. Conclusions-ROS generated by p22phox -based NADH/NADPH oxidase likely mediate the oxidative modification of LDL and might play a major role in pathogenesis of atherosclerotic coronary artery disease. (Arterioscler Thromb Vasc Biol.

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

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