Shinobu Imajoh-Ohmi

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

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.

The superoxide‐producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells

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)H‐dependent 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.

Involvement of NADH/NADPH Oxidase in Human Platelet ROS Production

Platelets play an important role in atherosclerotic and thromboembolic vascular diseases. It has been reported that reactive oxygen species (ROS) could modify platelet function, and platelets themselves have the ability to produce ROS. However, the enzymatic sources of ROS in platelets have not been fully determined. The NADH/NADPH oxidase system was originally identified as the major source of ROS in phagocytes. Recently, it has become evident that this oxidase is functionally expressed not only in phagocytes but also in various cell types. The present study was undertaken to test the hypothesis that NADH/NADPH oxidase might be expressed in human platelets. Lucigenin-enhanced chemiluminescence (L-CL) and electron spin resonance (ESR) method demonstrated that human platelets obtained from healthy volunteers released ROS, and the released ROS were increased by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore. Homogenates of human platelets, as well as MEG01 cells, megakaryocytic cell line, had the enzymatic activity to produce superoxide in NADH/NADPH-dependent manners. This enzymatic activity was suppressed by diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase. Western blot analysis demonstrated that platelets and MEG01 cells expressed p22(phox) and p67(phox) proteins, components of NADH/NADPH oxidase. Thus, human platelets have the enzymatic activity of p22(phox)-based NADH/NADPH oxidase, and this oxidase is likely one of the important sources of ROS in platelets.

Superoxide Generation in Directional Coronary Atherectomy Specimens of Patients With Angina Pectoris: Important Role of NAD(P)H Oxidase

Objective—NADH/NADPH oxidase is an important source of reactive oxygen species (ROS) in the vasculature. Recently, we demonstrated that p22phox, 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 p22phox, 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 p22phox 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 p22phox 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 p22phox 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.

Oxidative stress in the pathogenesis of thoracic aortic aneurysm : Protective role of statin and angiotensin II type 1 receptor blocker

OBJECTIVE The pathogenesis of thoracic aortic aneurysms (TAA) is still unclear. A recent investigation indicated that angiotensin II, a potent activator of NADH/NADPH oxidase, plays an important role in aneurysmal formation. We investigated the potential role of p22phox-based NADH/NADPH oxidase in the pathogenesis of TAA. METHODS Human thoracic aneurysmal (n=40) and non-aneurysmal (control, n=39) aortic sections were examined, and the localization of p22phox, an essential component of the oxidase, and its expressional differences were investigated by immunohistochemistry and Western blot. In situ reactive oxygen species (ROS) generation was examined by the dihydroethidium method, and the impact of medical treatment on p22phox expression was investigated by multiple regression analysis. RESULTS In situ production of ROS and the expression of p22phox increased markedly in TAA throughout the wall, and Western blot confirmed the enhanced expression of p22phox. The expression was more intense in the regions where monocytes/macrophages accumulated. In these inflammatory regions, numerous chymase-positive mast cells and angiotensin converting enzyme-positive macrophages were present. Their localization closely overlapped the in situ activity of matrix metalloproteinase and the expression of p22phox. Multiple regression analysis revealed that medical treatment with statin and angiotensin II type 1 receptor blocker (ARB) suppressed p22phox expression in TAA. CONCLUSION Our findings indicate the role of p22phox-based NADH/NADPH oxidase and the local renin-angiotensin system in the pathogenesis of TAA. Statin and ARB might have inhibitory effects on the formation of aneurysms via the suppression of NADH/NADPH oxidase.

Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of mu-calpain in human platelets.

The intracellular thiol protease μ-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit. Activation of μ-calpain requires calcium influx across the plasma membrane and the subsequent autoproteolytic conversion of the 80-kDa large subunit to a 78-kDa “intermediate” and a 76-kDa fully autolyzed form. Currently, there is limited information on the substrate specificities and functional roles of these distinct active forms of μ-calpain within the cell. Using antibodies that can distinguish among the 80-, 78-, and 76-kDa forms of μ-calpain, we have demonstrated a close correlation between the autolytic generation of the 78-kDa enzyme and the proteolysis of the non-receptor tyrosine phosphatase, PTP-1B, in ionophore A23187-stimulated platelets. Time course studies revealed that pp60c- src proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of μ-calpain (76 kDa). In vitroproteolysis experiments with purified μ-calpain and immunoprecipitated PTP-1B or pp60c- src confirmed selective proteolysis of pp60c- src by the 76-kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of μ-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of μ-calpain have demonstrated that the initial conversion of the μ-calpain large subunit to the 78-kDa form is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of μ-calpain (76 kDa) is responsible for the shedding of procoagulant-rich membrane vesicles from the cell surface. These studies demonstrate the existence of multiple active forms of μ-calpain within the cell, that have unique substrate specificities and distinct functional roles.

Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor alpha in human monoblast U937 cells.

Interferon‐γ (IFN‐γ), vitamin D3 (VD), and retinoic acid (RA) induce differentiation of human monoblastic leukemia U937 cells to macrophage‐like cells with potential superoxide anion‐generating activity upon further stimulation. Here we report that U93 7 cells thus differentiated show various responses to apoptotic induction with a cytotoxic anti‐Fas antibody and tumor necrosis factor (TNF). VD‐or RA‐treated U937 cells acquired resistance against Fas‐ or TNF receptor (TNFR)‐mediated apoptosis, whereas apoptotic cell death was accelerated in IFN‐γ‐treated cells. By flow cytometric analyses, no decrease in expression of surface Fas antigen or p55 TNFR was observed in differentiated U937 cells. Cell surface expression of CD11b was seen only when differentiation was induced with VD or RA but not with IFN‐γ. The growth of VD‐ or RA‐treated cells was retarded but IFN‐γ‐treated cells were prolific. These findings suggest that the differentiation state differs with the inducer and that the cellular response to apoptotic induction is closely related to the state including the cell cycle. J. Leukoc. Biol. 60: 778–783; 1996.

Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser502 that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.

Membrane Binding Properties and Terminal Residues of the Mature Hepatitis C Virus Capsid Protein in Insect Cells

ABSTRACT The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with α-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.