Lucia S. Yoshida

Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase.

ABSTRACT Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O2− generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67phox, p47phox, and p40phox, which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) βII rather than an effect of GT on PKC βII activity, suggesting a failure of PKC βII to associate with the substrate, p47phox, on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.

Expression of NADPH oxidases and enhanced H2O2-generating activity in human coronary artery endothelial cells upon induction with tumor necrosis factor-α

Tumor necrosis factor (TNF)-alpha, which potentiates reactive oxygen species (ROS) generation, is crucial for the development of coronary arteritis and aneurysm in Kawasaki disease. We hypothesized that vascular NADPH oxidase (Nox) enzymes participate in the TNF-alpha-triggered endothelial damage through elevating ROS generation. Thus, we herein examine the expression of Nox enzymes in human coronary artery endothelial cells (HCAEC) and the effects of TNF-alpha on Nox-mediated ROS generation. We show that HCAEC in culture spontaneously generate H(2)O(2) at basal level (0.53 nmol/min/mg protein). In searching for Nox components responsible for the H(2)O(2) generation, two distinct isoforms of Nox4 are found expressed in HCAEC: the prototype Nox4A and the shorter Nox4B, respectively in the postnuclear supernatant and the nuclear fractions. Other expressed Nox family components are: as mRNAs, Nox4C, Nox4D, Nox1, p51(nox), and Racs; as mRNAs and proteins, Nox2, p22(phox), p47(phox), and p67(phox). The H(2)O(2)-generating activity increases up to three-fold upon inclusion of TNF-alpha in culture, concomitantly with augmented expressions of Nox4A, p22(phox), p47(phox) and p67(phox) proteins. Together, these results suggest that Nox2 and Nox4A enzymes are induced by TNF-alpha endowing HCAEC with enhanced ROS-generating activity, which may play a role in the initial endothelial dysfunction through oxidative stress.

Mutation at Histidine 338 of gp91 phox Depletes FAD and Affects Expression of Cytochrome b 558 of the Human NADPH Oxidase

Defective NADPH oxidase components prevent superoxide (O⨪2) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91 phox subunit of cytochromeb 558 and usually lack gp91 phox protein completely (X910). gp91 phox is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O⨪2, but presented a diminished expression of gp91 phox containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67 phox and p47 phox was normal. However, the FAD content in his neutrophil membranes was as low as that of X910patients, suggesting complete depletion of FAD in his gp91 phox . This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91 phox in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient’s membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.

Fungal Metabolite Gliotoxin Targets Flavocytochrome b558 in the Activation of the Human Neutrophil NADPH Oxidase

ABSTRACT Fungal gliotoxin (GT) is a potent inhibitor of the O2−-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C βII with p47phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O2−-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b558 is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b558 and cytosolic components (native cytosol or recombinant p67phox, p47phox, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 μM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67phox, p47phox, and Rac2 with flavocytochrome b558 is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b558s ability to reconstitute O2− generation, supporting the conclusion at the cellular level. The flavocytochrome b558 heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b558, which is accessible only before oxidase activation.

Shikonin inhibits IgE-mediated histamine release by human basophils and Syk kinase activity

Abstract.Objective:Shikonin, a component of the herbal medicine “Shikon”, is known to suppress inflammatory reactions, but its molecular targets are not identified. This study examines the effect of shikonin on human basophil degranulation response and aims to identify its targets.Materials:Human basophils in isolated leukocytes from healthy volunteers’ peripheral blood; recombinant human Syk and Lyn tyrosine kinases. Methods:Histamine release from basophils stimulated with anti-IgE antibody was analyzed fluorimetrically. Syk and Lyn kinase activities were tested in Vitro with recombinant proteins and analyzed by off-chip mobility shift assay.Results:Shikonin dose-dependently inhibited the histamine release from basophils induced by anti-IgE antibody (IC50 = 2.6 ± 1.0 µM; mean ± SEM). A search for the target(s) of shikonin in the signal cascade of IgE-mediated activation showed that it strongly inhibits Syk (IC50 = 7.8 µM, in the recombinant kinase assay), which plays a pivotal role in the degranulation response. A less significant inhibition was found for Lyn, which phosphorylates FcεRI-βγ subunits and also Syk.Conclusions:These results indicate that the inhibition of Syk-dependent phosphorylation events might underlie the blocked histamine release from human basophils, thus contributing to the anti-inflammatory effects of shikonin.

Expression of a p67(phox) homolog in Caco-2 cells giving O(2)(-)-reconstituting ability to cytochrome b(558) together with recombinant p47(phox).

Human normal and transformed (Caco-2) colon tissues as well as guinea pig gastric mucosal cells express Nox1, which is a homolog of the phagocyte NADPH oxidase subunit, gp91(phox) of membrane-bound cytochrome b(558). It was reported that Nox1-transfection to NIH 3T3 cells could provide O(2)(-)-generating ability, independently of regulatory cytosolic factors (Rac2, p67(phox), and p47(phox)) that are obligatory in the phagocyte oxidase system. Here, we detected and sequenced a p67(phox) homolog in Caco-2 almost identical to the neutrophil sequence, except for three nucleotide substitutions, two of which changed lysines 181 and 328 to arginines. Investigation of its ability to support O(2)(-)-generation in cell-free reconstitution experiments combining with neutrophil cytochrome b(558) showed O(2)(-)-generation, provided that recombinant p47(phox) was added. This result demonstrates that the intrinsic p67(phox) homolog of Caco-2 was able to function as a phagocyte p67(phox) for cytochrome b(558). The requirement of p47(phox) addition suggested that this component was absent in Caco-2 cells. Caco-2 membranes, used as a source of Nox1 in place of cytochrome b(558), did not show significant O(2)(-)-generation, which was mainly explained by their very little Nox1 expression.

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca2+ fluxes

Shikonin, an anti-inflammatory compound of “Shikon”, inhibits the neutrophil superoxide (O2•−) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca2+ concentration ([Ca2+]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca2+]i (by fluorescence) and O2•− generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca2+]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and store-operated Ca2+ entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca2+]i and O2•− generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca2+ fluxes showed that shikonin inhibits IICR and IICR-linked O2•− generation (IC50 values: 0.28 and 0.31 µM for [Ca2+]i and O2•−, respectively), as well as SOCE and SOCE-linked O2•− generation (IC50 values: 0.39 and 0.25 µM for [Ca2+]i and O2•−, respectively). These results suggested that shikonin inhibits the O2•− generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca2+ fluxes such as IICR and SOCE.

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.