Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O 2 activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys 423 , Lys 620 , and Lys 660 were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys 423 , Lys 620 , and Lys 660 , which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.

Background Severe asthma may involve both innate and Type-2 cytokine associated adaptive immunity. While IL-27 has been reported to potentiate Th1 responses (including the chemokine CXCL9) and suppress Th2 responses, its function in asthma is unknown. Objective Evaluate IL-27 expression in human asthma, alone and in combination with Type-2 immunity to determine the relationship to disease severity and CXCL9 expression. Model these interactions in vitro in human bronchial epithelial cells (HBEC). Methods Bronchoalveolar lavage (BAL) cells from 87 participants were evaluated for IL-27 mRNA and protein, alone and in association with epithelial CCL26 (a marker of Type-2 activation) in relation to asthma severity and CXCL9 mRNA. HBECs cultured in air liquid interface (ALI) and stimulated with IL-27 (1–100 ng/ml) with/without IL-13 (1 ng/ml) were evaluated for CXCL9 expression by qRT-PCR and ELISA. Phosphorylated and total STAT1/3 were detected by western blot. siRNA knockdown of STAT1 or STAT3 was performed. Results BAL cell IL-27 mRNA and protein were increased in asthma. Patients with evidence for Type-2 pathway activation had higher IL-27 expression (p=0.02). Combined IL-27 and CCL26 expression associated with more severe asthma and higher CXCL9 expression (p=0.004, 0.007 respectively), while IL-27 alone was associated with milder disease. In vitro, IL-13 augmented IL-27 induced CXCL9 expression which appeared to be due to augmented STAT1 activation and reduced STAT3 activation. Conclusions IL-27, in combination with a Type-2/CCL26 signature identifies a more severe asthma phenotype, perhaps through combined effects of IL-27 and IL-13 on STAT signaling. Understanding these interactions could lead to new targets for asthma therapy.

While the binding of biotin by streptavidin does not appear to be cooperative in the traditional sense of altered binding strength, it has been suggested that it may be cooperative in terms of differential structural changes in the protein. In this work we present intrinsic tryptophan fluorescence data as evidence of a cooperative structural change. The technique involves examination of the differences in fluorescence emission corresponding to distinct tryptophan populations accompanying protein-ligand binding. Specifically we note that the 335 nm emission population (i.e. more hydrophobic) saturates prior to the saturation of the 350 nm emission population commonly used in the standard binding activity assay. We also note that the wavelength of maximum emission, total integrated fluorescence emission and full width at half maximum during the titration of ligand into streptavidin also reach saturation before the expected 4:1 stoichiometric end point. This suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand.