Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention.

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65 °C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

Rab27a and Rab27b regulate neutrophil azurophilic granule exocytosis and NADPH oxidase activity by independent mechanisms.

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Signalling mechanisms for Toll-like receptor-activated neutrophil exocytosis: key roles for interleukin-1-receptor-associated kinase-4 and phosphatidylinositol 3-kinase but not Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF).

Lipopolysaccharide (LPS) stimulates exocytosis in neutrophils. The signalling molecules involved in the regulation of this mechanism are currently unknown. Using neutrophils from interleukin-1-receptor-associated kinase (IRAK)-4- and Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-deficient mice, we dissected the signalling pathways that control exocytosis. We analysed exocytosis of peroxidase-negative and azurophilic granules by following the mobilization of the beta2-integrin subunit CD11b and myeloperoxidase (MPO)-containing granules, respectively. IRAK-4-null neutrophils showed marked defects in both peroxidase-negative and azurophilic granule exocytosis in response to LPS. In contrast, the exocytic response to LPS of TRIF-deficient neutrophils was not different from that of wild-type cells. No differences were observed in the exocytosis of secretory organelles between IRAK-4-null and wild-type neutrophils when they were stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). Electron microscopy analysis showed that no morphological abnormalities were present in the granules of IRAK-4-deficient neutrophils, suggesting that the lack of exocytic response to LPS is not attributable to developmental abnormalities. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase (p38MAPK) is essential for the exocytosis of all neutrophil secretory organelles in response to LPS. Interestingly, we found that phosphatidylinositol 3-kinase (PI3K) is essential for azurophilic granule exocytosis but not for the mobilization of other neutrophil granules in response to LPS. Azurophilic granule exocytosis in response to Listeria monocytogenes was dependent on PI3K but not IRAK-4 activity, suggesting that alternative signalling pathways are activated in IRAK-4-deficient neutrophils exposed to whole bacteria. Our results identified IRAK-4, p38MAPK and PI3K as important regulatory components with different roles in the signalling pathways that control Toll-like receptor ligand-triggered neutrophil exocytosis.

DNase I inhibits a late phase of reactive oxygen species production in neutrophils.

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.

The Rab27a effectors JFC1/Slp1 and Munc13-4 regulate exocytosis of neutrophil granules.

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.

Cross-talk between IRAK-4 and the NADPH oxidase.

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes.

BACKGROUND: Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. RESULTS: Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. CONCLUSION: We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.