Endothelial Nitric Oxide Synthase is Regulated by ERK Phosphorylation at Ser602

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2–3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets

Seasonal influenza vaccination of healthcare workers : Systematic review of qualitative evidence

Background Most countries recommend that healthcare workers (HCWs) are vaccinated seasonally against influenza in order to protect themselves and patients. However, in many cases coverage remains low. A range of strategies have been implemented to increase uptake. Qualitative evidence can help in understanding the context of interventions, including why interventions may fail to achieve the desired effect. This study aimed to synthesise evidence on HCWs’ perceptions and experiences of vaccination for seasonal influenza. Methods Systematic review of qualitative evidence. We searched MEDLINE, EMBASE and CINAHL and included English-language studies which reported substantive qualitative data on the vaccination of HCWs for seasonal influenza. Findings were synthesised thematically. Results Twenty-five studies were included in the review. HCWs may be motivated to accept vaccination to protect themselves and their patients against infection. However, a range of beliefs may act as barriers to vaccine uptake, including concerns about side-effects, scepticism about vaccine effectiveness, and the belief that influenza is not a serious illness. HCWs value their autonomy and professional responsibility in making decisions about vaccination. The implementation of interventions to promote vaccination uptake may face barriers both from HCWs’ personal beliefs and from the relationships between management and employees within the targeted organisations. Conclusions HCWs’ vaccination behaviour needs to be understood in the context of HCWs’ relationships with each other, with management and with patients. Interventions to promote vaccination should take into account both the individual beliefs of targeted HCWs and the organisational context within which they are implemented

Weak Interactions between <i>Salmonella enterica</i> FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics

<div><p>The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhB_C) and other export apparatus proteins including FliK, FlhA_C and FliI. FliK and FlhA_C bound with micromolar affinity. K_D for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.</p></div

FlhA_C-FlhB_C binding.

<p>Ligand FlhA_C was exposed to 2, 1, 0.5, 0.25 and 0.125 μM FlhB_C. A, association with fits to a one-state model B, dissociation with fits to a global two-state model C, steady state analysis. D, k_obs vs. [FlhB_C] to estimate kinetic constants, R² = 0.98.</p

MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of L-arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c-Jun N-terminal kinase (JNK1 ), p38α, and ERK2 were characterized using optical biosensing with full length NOS3 and NOS3 specific peptides and phospho-peptides. Like p38α and ERK2, JNK1 exhibited high affinity binding to full length NOS3 (K 15 nM). Rate constants exhibited fast-on, slow-off binding (k = 4106 M s ; k = 6.2 x 10 s ). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1 the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633)

Kinetic constants determined from BLI experiments for binding to FlhB_C.

<p>ND, not determined. Constants are expressed with respect to the monomer concentrations.</p><p>Kinetic constants determined from BLI experiments for binding to FlhB_C.</p

FlhB_C-FliI binding.

<p>FlhB_C was used as ligand to examine binding of FliI in the absence and presence of ATP. A, Binding of 4, 2, 1, 0.5 and 0.25 μM FliI with fits to a global one-state association-then-dissociation model shown in red. The green points are 4μM FliI exposed to a sensor without FlhB_C, e.g. NSB. B, FlhB_C-FliI binding with FliI preequilibrated with 5 mM ATP and 5 mM MgCl₂ added to all phases. FliI concentrations were 2, 1, 0.5, 0.25, 0.13, 0.062 and 0 μM. Green points represent the 2 uM sample binding to sensor without ligand. C, Steady state analydis of FliI with ATP from panel B, K_D = 1.1 μM.</p

FlhB_C kinetic survey.

<p>Purified T3S proteins were biotinylated and used as ligands. All analyte concentrations were 1 μM. Association and dissociation phases were 180 seconds in all cases. A, FlhB_C analyte binding to ligands FlhA_C (red), FlhB_C (blue), FliH (green), FliI (orange), FliJ (magenta) and FliK (black). B, nonspecific binding control of analyte apparatus proteins binding to sensor-tethered biotinylated BSA. Analyte colors are the same as ligand colors in A. C, Analyte apparatus proteins binding to ligand FlhB_C. Colors are the same as in B.</p

Parameters determined by simulation of FliK-FlhB_C binding.

<p>Parameters determined by simulation of FliK-FlhB_C binding.</p

Simulation of FliK-FlhB_C binding.

<p>A and B, association and dissociation phases. Concentrations of FlhB_C were 5, 4, 3, 2, 1, 0.5, 0.25, and 0 μM. The 0.5 and 0.25 μM traces are unlabeled. Fits to individual two-state exponentials are shown as red lines. C-G, Simulations of the 5, 4, 3, 2 and 1 μM data with a conformational change model using global parameters for k_off and k_on and k_off for transition to the conformationally altered state (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134884#pone.0134884.t002" target="_blank">Table 2</a>). H, Apparent k_on vs [FlhB_C] to determine the global k_on (= slope).</p