Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

Glucose and lipid metabolism alterations in liver and adipose tissue pre-dispose p47phox knockout mice to systemic insulin resistance

Oxidative stress due to enhanced production or reduced scavenging of reactive oxygen species (ROS) has been associated with diet (dyslipidemia) induced obesity and insulin resistance (IR). The present study was undertaken to assess the role of p47^phox in IR using wild type (WT) and p47^phox?/? mice, fed with different diets (HFD, LFD or Chow). Augmented body weight, glucose intolerance and reduced insulin sensitivity were observed in p47^phox?/? mice fed with 45% HFD and 10% LFD. Further, body fat and circulating lipids were increased significantly with 5 weeks LFD feeding in p47^phox?/? mice, while parameters of energy homeostasis were reduced as compared with WT mice. LFD fed knockout (KO) mice showed an enhanced hepatic glycogenolysis, and reduced insulin signalling in liver and adipose tissue, while skeletal muscle tissue remained unaffected. A significant increase in hepatic lipids, adiposity, as well as expression of genes regulating lipid synthesis, breakdown and efflux were observed in LFD fed p47^phox?/? mice after 5 weeks. On the other hand, mice lacking p47^phox demonstrated altered glucose tolerance and tissue insulin sensitivity after 5 weeks chow feeding, while changes in body weight, respiratory exchange ratio (RER) and heat production are non-significant. Our data demonstrate that lack of p47^phox is sufficient to induce IR through altered glucose and lipid utilization by the liver and adipose tissue.     

Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by γ-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.     

Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22^phox, NOX4, and p67^phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22^phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22^phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22^phox, NOX4, p67^phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.     

NOX in liver fibrosis

NADPH oxidase is a multi-protein complex producing reactive oxygen species (ROS) both in phagocytic cells, being essential in host defense, and in non-phagocytic cells, regulating intracellular signalling. In the liver, NADPH oxidase plays a central role in fibrogenesis. A functionally active form of the NADPH oxidase is expressed not only in Kupffer cells (phagocytic cell type) but also in hepatic stellate cells (HSCs) (non-phagocytic cell type), suggesting a role of the non-phagocytic NADPH oxidase in HSC activation. Consistent with this concept, profibrogenic agonists such as Angiotensin II (Ang II) and platelet derived growth factor (PDGF), or apoptotic bodies exert their activity through NADPH oxidase-activation in HSCs. Both pharmacological inhibition with DPI and genetic studies using p47(phox) knockout mice provided evidence for a central role of NADPH oxidase in the regulation of HSC-activity and liver fibrosis. In addition to the p47(phox) component, only Rac1 has been identified as a functional active component of the NADPH oxidase complex in HSCs.     

Conformational changes in p47 upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis

The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47^phox promotes its translocation with p67^phox and p40^phox, followed by membrane interaction and assembly with flavocytochrome b₅₅₈ into a functional complex. To characterise p47^phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47^phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain.     

高糖刺激下PKC/NADPH氧化应激途径对内皮细胞活性氧生成的影响及其机制探讨

目的:探讨高糖刺激下PKC/NADPH氧化应激途径对内皮细胞活性氧生成的影响。方法:实验分为正常对照组、NaOH对照组、DMSO对照组、20 mM葡萄糖处理4小时组(高糖组)、1 mol佛波酯预处理0.5小时再加入20 m M葡萄糖处理组(佛波酯组)和4 mol金丝桃素预处理0.5小时再加入20 mM葡萄糖处理组(金丝桃素组);Ⅱ型胶原酶消化法分离人脐静脉内皮细胞;流式细胞术检测细胞内活性氧;免疫荧光检测内皮细胞Ⅷ因子和NADPH氧化酶亚基p47phox定位。结果:1与正常对照组相比,高糖组细胞内活性氧增高(P0.05,n=3),与正常对照组和高糖组相比,佛波酯组HUVECs内活性氧的产生显著增加(P0.05,n=3),与正常对照组和高糖组相比,金丝桃素组HUVECs内活性氧的产生明显减少(P0.05,n=3);2正常对照组和金丝桃素组中细胞NADPH氧化酶亚基p47phox主要位于胞浆,而佛波酯组和高糖组的NADPH氧化酶亚基p47phox位于胞膜。结论:高糖通过PKC信号通路调节内皮细胞NADPH氧化酶亚基p47phox的移位从而增加细胞内活性氧的生成。     

Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.     

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.     

Interaction between the SH3 domains and C-terminal proline-rich region in NADPH oxidase organizer 1 (Noxo1)

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1.     

Essential role of Rac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression

Nerve growth factor (NGF) regulates the nociceptive properties of a subset of small diameter sensory neurons by increasing the expression of the heat-sensing transient receptor potential (TRP) channel, TRPV1. This action involves activation of the tyrosine kinase receptor (Trk) A/p38 MAPK pathway. Recent studies indicate that activation of TrkA promotes superoxide generation via NADPH oxidase. In this study, we determined whether the NADPH oxidase pathway is involved in NGF-stimulated TRPV1 expression using a rat pheochromocytoma 12 line and rat dorsal root ganglion neurons. Treatment of these cells with NGF (100 ng/mL) increased TRPV1 protein expression (approx. twofold) but not mRNA. This increase was mimicked by H(2)O(2) and attenuated by catalase and inhibitors of NADPH oxidase. NGF stimulated NADPH oxidase activity, while 24 h exposure further increased expression of the Rac1 and gp91(phox) subunits of the holoenzyme. Inhibition of NADPH oxidase by transient transfection of a dominant negative Rac1 mutant (RacN17) plasmid blocked NGF-stimulated TRPV1 protein expression, while expression of a constitutively active Rac1 increased basal and NGF-stimulated TRPV1 levels. Inhibition of NADPH oxidase activity also attenuated NGF-dependent p38 MAPK activation. We conclude that the Rac1/NADPH oxidase pathway regulates p38 activation and TRPV1 expression which aids in the maintenance of peripheral neuron integrity and pain perception.     

The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies

Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of (15)N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database (), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.     

A new chronological survival assay in mammalian cell culture

c-Abl is activated by oxidative stress but its precise function in cell response to this stress is elusive. Studies of c-Abl-/- osteoblasts revealed that c-Abl played a negative role in the induction of peroxiredoxin I (Prx I, Prdx I), an anti-oxidant protein with tumor suppression activity, but it provided a protective role against oxidative stress. In contrast, Atm, a signaling molecule that interacts with c-Abl and is required for c-Abl activation, served a totally different function. The significance of these findings is discussed here in the context of aging and tumorigenesis and their links to reactive oxygen species.     

Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation

Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.     

Intracellular Ca can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells

The aim of the present study is to determine the role of intracellular Ca²⁺ in VEGF signaling. We demonstrate that reduction in Ca²⁺ by chelating compound BAPTA-AM or by IP₃-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca²⁺. These results suggest an essential role for Ca²⁺ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.