Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 µg/mL-azithromycin and 10 and 50 µg/mL-chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.

TAT cell-penetrating peptide modulates inflammatory response and apoptosis in human lung epithelial cells.

Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.

The origin of C1A-C2 interdomain interactions in protein kinase Calpha.

The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.

Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy.

In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 microm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.

Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell.

The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D.

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.

Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.

Structural basis of binding of high-affinity ligands to protein kinase C: prediction of the binding modes through a new molecular dynamics method and evaluation by site-directed mutagenesis.

The structural basis of protein kinase C (PKC) binding to several classes of high-affinity ligands has been investigated through complementary computational and experimental methods. Employing a recently developed q-jumping molecular dynamics (MD) simulation method, which allows us to consider the flexibility of both the ligands and the receptor in docking studies, we predicted the binding models of phorbol-13-acetate, phorbol-12,13-dibutyrate (PDBu), indolactam V (ILV), ingenol-3-benzoate, and thymeleatoxin to PKC. The "predicted" binding model for phorbol-13-acetate is virtually identical to the experimentally determined binding model for this ligand. The predicted binding model for PDBU is the same as that for phorbol-13-acetate in terms of the hydrogen-bonding network and hydrophobic contacts. The predicted binding model for ILV is the same as that obtained in a previous docking study using a Monte Carlo method and is consistent with the structure-activity relationships for this class of ligands. Together with the X-ray structure of phorbol-13-acetate in complex with PKCdelta C1b, the predicted binding models of PDBu, ILV, ingenol-3-benzoate, and thymeleatoxin in complex with PKC showed that the binding of these ligands to PKC is governed by a combination of several highly specific and optimal hydrogen bonds and hydrophobic contacts. However, the hydrogen-bonding network for each class of ligand is somewhat different and the number of hydrogen bonds formed between PKC and these ligands has no correlation with their binding affinities. To provide a direct and quantitative assessment of the contributions of several conserved residues around the binding site to PKC-ligand binding, we have made 11 mutations and measured the binding affinities of the high-affinity PKC ligands to these mutants. The results obtained through site-directed mutagenic analysis support our predicted binding models for these ligands and provide new insights into PKC-ligand binding. Although all the ligands have high affinity for the wild-type PKCdelta C1b, our site-directed mutagenic results showed that ILV is the ligand most sensitive to structural perturbations of the binding site while ingenol-3-benzoate is the least sensitive among the four classes of ligands examined here. Finally, we have employed conventional MD simulations to investigate the structural perturbations caused by each mutation to further examine the role played by each individual residue in PKC-ligand binding. MD simulations revealed that several mutations, including Pro11 --> Gly, Leu21 --> Gly, Leu24 --> Gly, and Gln27 --> Gly, cause a rather large conformational alteration to the PKC binding site and, in some cases, to the overall structure of the protein. The complete abolishment or the significant reduction in PKC-ligand binding observed for these mutants thus reflects the loss of certain direct contacts between the side chain of the mutated residue in PKC and ligands as well as the large conformational alteration to the binding site caused by the mutation.

Synthesis and tumor-promoting activities of 12-Epi-phorbol-12,13-dibutyrate.

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O2-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the beta-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.

Protein kinase C ligands based on tetrahydrofuran templates containing a new set of phorbol ester pharmacophores.

A series of substituted tetrahydrofurans with an embedded glycerol backbone carrying additional tetrahydrofuranylideneacetate or tetrahydrofuranylacetate motifs were grouped into four distinct templates (I-IV) according to stereochemistry. The compounds were designed to mimic three essential pharmacophores (C(3)-C=O, C(20)-OH and C(13)-C=O) of the phorbol esters according to a new, revised model. The tetrahydrofuran ring was constructed from glycidyl 4-methoxyphenyl ether, and the structures of the isomeric templates were assigned by NMR spectroscopy, including NOE. The binding affinity for protein kinase C (PKC) was assessed in terms of the ability of the ligands to displace bound [(3)H-20]phorbol 12, 13-dibutyrate (PDBU) from a recombinant alpha isozyme of PKC. Geometric Z- and E-isomers (1 and 3, respectively) containing a tetrahydrofuranylideneacetate motif were the most potent ligands with identical K(i) values of 0.35 microM. Molecular modeling studies of the four templates showed that the rms values when fitted to a prototypical phorbol 12,13-diacetate ester correlated inversely with affinities in the following order: I approximately II > III > IV. These compounds represent the first generation of rigid glycerol templates seeking to mimic the binding of the C(13)-C=O of the phorbol esters. The binding affinities of the most potent compounds are in the same range of the diacylglycerols (DAGs) despite the lack of a phorbol ester C(9)-OH pharmacophore surrogate. This finding confirms that mimicking the binding of the C(13)-C=O pharmacophore of phorbol is a useful strategy. However, since the C(9)-OH and C(13)-C=O in the phorbol esters appear to form an intramolecular hydrogen bond that functions as a combined pharmacophore, it is possible the lack of this combined motif in the target templates restricts the compounds from reaching higher binding affinities.

Synthesis, computer modeling and biological evaluation of novel protein kinase C agonists based on a 7-membered lactam moiety.

4-Hydroxymethyl-5a-methyl-1,3,4,5,5a beta,6,7,8,9,9a alpha-decahydro-2H-benz[d]azepin-2-ones (4-12), which were designed to mimic the biologically active conformation of teleocidins and benzolactams, were synthesized and evaluated for the ability to compete with [3H]phorbol 12,13-dibutyrate in a PKC delta binding assay. Among the compounds, 10-12 showed potent binding affinity, with inhibition constants (Ki) of low nanomolar order. Computational docking simulation also indicates that the relative positions of the hydrogen-bonding sites and hydrophobic regions of the compounds are well matched to the PKC delta binding site.

NMR structure of a protein kinase C-gamma phorbol-binding domain and study of protein-lipid micelle interactions.

Classical protein kinase C (PKC) family members are activated by the binding of various ligands to one of several cysteine-rich domains of the enzyme. The natural agonist, diacylglycerol (DAG), and the natural product superagonist, phorbol dibutyrate (PDB), activate the enzyme to produce wide-ranging physiological effects. The second cysteine-rich (Cys2) domain of rat brain PKC-gamma was expressed and labeled with 15N and 13C, and the solution structure was determined to high resolution using multidimensional heteronuclear NMR methods. The phorbol binding site was identified by titrating this domain with phorbol-12,13-dibutyrate (PDB) in the presence of organic cosolvents. Titrations of this domain with lipid micelles, in the absence and presence of phorbols, indicate selective broadening of some resonances. The observed behavior indicates conformational exchange between bound and free states upon protein-micelle interaction. The data also suggest that half of the domain, including the phorbol site and one of the zinc sites, is capable of inserting into membranes.