Flotillins Interact with PSGL-1 in Neutrophils and,upon Stimulation,Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

Background

Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation.

Methodology/Principal Findings

We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform.Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1.

Conclusions/Significance

These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells

We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity.     

Proteolytic cleavage of annexin 1 by human leukocyte elastase

Annexin 1 has been shown to participate through its unique N-terminal domain in the recruitment and activation of leukocytes at sites of inflammation. Peptides derived from this domain are true mimetics of the annexin 1 action in all inflammation models tested and most likely serve as the active entities generated at sites of inflammation. To elucidate mechanisms underlying peptide generation we used isolated blood leukocytes and endothelial cell monolayers. We show that following endothelial adhesion, annexin 1 was externalized from leukocytes and rapidly cleaved. Addition of purified annexin 1 to degranulating leukocytes resulted in the truncation of annexin 1, which seemed to depend on the proteolytic activity of human leukocyte elastase (HLE). The capacity of elastase to proteolytically cleave annexin 1 was confirmed using both purified annexin 1 and HLE. The identification of annexin 1 as a substrate for HLE supports the model in which annexin 1 participates in regulating leukocyte emigration into inflamed tissue through N-terminal peptides generated at inflammatory sites.     

Characterization of the East Asian Variant of Aldehyde Dehydrogenase-2: BIOACTIVATION OF NITROGLYCERIN AND EFFECTS OF Alda-1*

The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at ≥5 mm. In the presence of 1 mm NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.     

Identification of the bacterial superoxide dismutase (SodM) as plant-inducible elicitor of an oxidative burst reaction in tobacco cell suspension cultures

Three of the most abundant proteins (OmpW, MopB and SodM) of the extracellular proteome of Xanthomonas campestris pv. campestris were analysed in a luminol-based oxidative burst assay to identify novel pathogen-associated molecular patterns (PAMP). Tobacco cell suspension cultures were used as a model system to monitor elicitor induced plant defence reaction. The candidate proteins were isolated from two-dimensional gels prior to application to the oxidative burst assay. The superoxide dismutase (SodM) was the only isolated protein that could elicit a notable hydrogen peroxide (H2O2) production in tobacco cell cultures indicating the initiation of plant defence. An alignment of the SodM sequences from X. campestris pv. campestris and Escherichia coli revealed 55.7% identity and 29% of the sequence were substitutions for amino acids with similar physico-chemical properties. By using a commercially available purified E. coli derived SodM preparation, it was possible to show that the amino acid sequence of this protein is responsible for the elicitation of an oxidative burst reaction in the tobacco cell culture model. This suggests that the bacterial superoxide dismutase is a novel pathogen-associated molecular pattern. The minimal elicitor active sequence, however, is still elusive.     

Physiology of the esophageal pressure transition zone: separate contraction waves above and below

Manometrically measured peristaltic pressure amplitude displays a well-defined trough in the upper esophagus. Whereas this manometric "transition zone" (TZ) has been associated with striated-to-smooth muscle fiber transition, the underlying physiology of the TZ and its role in bolus transport are unclear. A computer model study of bolus retention in the TZ showed discoordinated distinct contraction waves above and below. Our aim was to test the hypothesis that distinct upper/lower contraction waves above/below the manometric TZ are normal physiology and to quantify space-time coordination between tone and bolus transport through the TZ. Eighteen normal barium swallows were analyzed in 6 subjects with concurrent 21-channel high-resolution manometry and digital fluoroscopy. From manometry, the TZ center (nadir pressure amplitude) and the upper/lower margins of the pressure trough were objectively quantified. Using fluoroscopy, we quantified space-time trajectories of the bolus tail and bolus tail pressures and maximum intraluminal pressures proximal to the tail with their space-time trajectories. In every swallow, the bolus tail followed distinct trajectories above/below the TZ, separated by a well-defined spatial "jump" that terminated an upper contraction wave and initiated a lower contraction wave (3.32 +/- 1.63 cm, P = 0.0004). An "indentation wave" always formed within the TZ distal to the upper wave, increasing in amplitude until the lower wave was initiated. As the upper contraction wave tail entered the TZ, it slowed and the tail pressure reduced rapidly, while indentation wave pressure increased to normal tail pressure values at the initiation of the lower wave. The TZ was a special zone of segmental contraction. The TZ is, physiologically, the transition from an upper contraction wave originating in the proximal striated esophagus to a lower contraction wave that moves into the distal smooth muscle esophagus. Complete bolus transport requires coordination of upper/lower waves and sufficient segmental squeeze to fully clear the bolus from the TZ during the transition period.     

Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin

Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd.