S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Protein engineering to develop a redox insensitive endothelial nitric oxide synthase.

The zinc tetrathiolate (ZnS4) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H2O2). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H2O2. The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

A novel pathway for human endothelial cell activation by antiphospholipid/anti-ß2 glycoprotein I antibodies.

Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly ß(2)GPI, and activate endothelial cells (ECs) in a ß(2)GPI-dependent manner after binding of ß(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-ß(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-ß(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-ß(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-ß(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

eNOS activation and NO function: structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity.

Rather than being a constitutive enzyme as was first suggested, endothelial nitric oxide synthase (eNOS) is dynamically regulated at the transcriptional, posttranscriptional, and posttranslational levels. This review will focus on how changes in eNOS function are conferred by various posttranslational modifications. The latest knowledge regarding eNOS targeting to the plasma membrane will be discussed as the role of protein phosphorylation as a modulator of catalytic activity. Furthermore, new data are presented that provide novel insights into how disruption of the eNOS dimer prevents eNOS uncoupling and the production of superoxide under conditions of elevated oxidative stress and identifies a novel regulatory region we have termed the 'flexible arm'.

Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption.

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

Beta-actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme.

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase C delta.

Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H2O2 levels. The increases in H2O2 were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H2O2, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCdelta activity in response to shear stress, and the overexpression of PKCdelta attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCdelta overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H2O2. Together, our data indicate that shear stress decreases PKCdelta activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H2O2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.

Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae).

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q. gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed.

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis. RESULTS: We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis. CONCLUSION: Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.