Effects of antimicrobial peptides of neutrophils on tumor and normal cells in culture

We performed a comparative study of effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine bactenecin 5 (Bac5) on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic beta-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic properties. Bac5 belongs to the group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a non-lytic mechanism. We have shown that while PG1 exerts distinct and fast cytotoxic effects on most of used tumor cells being slightly less toxic for nontransformed host cell, the proline-rich Bac5 is much less cytotoxic for all the cells tested. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as had been previously established for human defensins by Panyutich et al., 1995). Pre-incubation of PG1 with alpha1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of alpha1-antitrypsin was reduced after interaction of the serpin with PG1 (not with Bac5). Confocal microscopy experiments allowed to monitor the internalization of fluorescent labeled (by BODIPY FL) peptides into target cells and their intracellular distribution. Bac5-BODIPY (at 5 microM) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations was also able to enter the cells without significant damage to them. The comparative study of the kinetics of the peptides uptake into the target cells and the influence of low temperature, energy-depletion and endocytosis inhibitors on the process of the internalization of the peptides into the cells was carried out using flow cytometry.     

Role of Cathelicidin Peptides in Bovine Host Defense and Healing

The in vitro antimicrobial activities and biological effects on host cells were compared for the bovine cathelicidins BMAP-28, an alpha-helical AMP, and Bac5 and Bac7, proline-rich AMPs. Our results confirm that the broad-spectrum activity of BMAP-28 correlates with a high capacity to interact with and permeabilize bacterial membranes, whereas the proline-rich AMPs selectively internalize into the cytoplasm of susceptible Gram-negative bacteria with a non-lytic mechanism. All peptides efficiently translocated into mammalian fibroblastic cells, but while Bac5 and Bac7(1–35) localized to nuclear structures and induced cellular proliferation, BMAP-28 associated with mitochondria and did not induce proliferation. Moreover, BMAP-28 was considerably more cytotoxic than the proline-rich peptides due to cytolytic and pro-apoptotic effects. Our results highlight important functional differences among the bovine cathelicidins and suggest that they contribute to an integrated response of the host to infection, with distinct but complementary activities.     

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

ChBac3.4: A Novel Proline-Rich Antimicrobial Peptide from Goat Leukocytes

We isolated a new proline-rich peptide, ChBac3.4, from leukocytes of the goat (Capra hirca) and determined its amino acid sequence by Edman degradation and mass spectrometry. ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL–NH2) had over 50% sequence identity to the Bac5 peptides found in the leukocytes of goats, sheep and cattle. ChBac3.4 exhibited broadspectrum antimicrobial activity, especially under low salt conditions. Since E. coli ML35p treated with ChBac3.4 manifested increased outer and inner membrane permeability and a rapid and extensive loss of cytoplasmic potassium, the antimicrobial properties of this peptide may depend, in part, on its ability to damage microbial membranes. Nevertheless, even high concentrations of ChBac3.4 were not significantly hemolytic for human erythrocytes. In vitro, ChBac3.4 was selectively cytotoxic, damaging human K562 erythroleukemia cells and human U937 hystiocytic lymphoma cells, but not other human target cells. ChBac3.4 appears to differ from other proline-rich cathelicidins in virtue of its increased ability to damage microbial membranes. This novel antimicrobial peptide warrants further study, especially with respect to its various effects on microbial and mammalian cells. An erratum to this article can be found at     

Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides

In contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.     

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

Regulation of Endoribonuclease Activity of Alpha-Type Proteasome Subunits in Proerythroleukemia K562 Upon Hemin-Induced Differentiation

The proteasome is the main intracellular proteolytic machine involved in the regulation of numerous cellular processes, including gene expression. In addition to their proteolytic activity, proteasomes also exhibit ATPase/helicase (the 19S particle) and RNAse (the 20S particle) activities, which are regulated by post-translational modifications. In this report we uncovered that several 20S particle subunits: α1 (PSMA6), α2 (PSMA2), α4 (PSMA7), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3) possess RNAse activity against the p53 mRNA in vitro. Furthermore, we found that the RNAse activity of PSMA1 and PSMA3 was regulated upon hemin-induced differentiation of K562 proerythroleukemia cells. The decrease in RNAse activity of PSMA1 and PSMA3 was paralleled by changes in their status of phosphorylation and ubiquitylation. Collectively, our data support the notion that proteasomal RNAse activity may be functionally important and provide insights into the potential mechanism of p53 repression in erythroleukemia cells by RNAse activity of the 20S α-type subunits.     

Inhibition of heat- and chemical-induced aggregation of various proteins reveals chaperone-like activity of the acute-phase component and serine protease inhibitor human α1-antitrypsin

In vitro chaperone-like activity of the serpin family member and plasma acute-phase component human α₁-antitrypsin (AAT) has been shown for the first time. Results of light-scattering experiments demonstrated that AAT efficiently inhibits both heat- and chemical-induced aggregation of various test proteins including alcohol dehydrogenase, aldolase, carbonic anhydrase, catalase, citrate synthase, enolase, glutathione S-transferase, l-lactate dehydrogenase, and β_L-crystallin. The results suggest that the unique metastable serpin architecture enables dual function, protease inhibiton as well as chaperone activity and highlight the serpin superfamily as a possible source of additional intra- and extracellular chaperones (e.g. α₁-antichymotrypsin). The present finding is surprising in the light of the well-known role of mutated forms of AAT and other serpins in the pathogenesis of diseases called serpinopathies that featured with aberrant conformational transitions and consequent self-aggregation of serpin proteins.     

α-Synuclein promotes clathrin-mediated NMDA receptor endocytosis and attenuates NMDA-induced dopaminergic cell death

Abnormalities of α-synuclein (α-syn) and NMDA receptors (NMDARs) are implicated in the pathogenesis of Parkinson's disease. However, how these proteins interact with each other has not been elucidated. Here, the effect of α-syn on NMDARs was investigated by examining the alterations of surface NMDAR NR1 subunits in MES23.5 dopaminergic cells transfected with the human α-syn gene as well as in cells treated with extracellularly added human α-syn. As demonstrated previously that α-syn can enter cells in a non-endocytic manner without being degraded by the cellular proteolytic systems, the extracellularly added α-syn entered the cytoplasm of MES23.5 cells in a concentration-dependent manner. Both the α-syn-transfected cells and α-syn-treated cells exhibited increased intracellular α-syn levels and reduced surface NR1 without altering the total NR1. The α-syn-induced surface NR1 reduction was accompanied by suppression of NMDA-elicited intracellular Ca(2+) elevation and reductions of NMDA-induced caspase 3 activation and cell death, which was abolished by hypotonic shock and K(+) depletion, a procedure that blocks clathrin-mediated endocytosis, and by suppression of RAB5B expression with anti-RAB5B oligonucleotides. The data obtained provide evidence for the first time that α-syn may promote clathrin-mediated NMDAR endocytosis.     

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and β-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca2+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1-B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by D-galactose, α-lactose, lactulose, LacNAc, thiodigalactoside (TDG), L-fucose, D-mannose, D-glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca2+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

A fluorescent probe of polyamine transport accumulates into intracellular acidic vesicles via a two-step mechanism

Mammalian polyamine carriers have not yet been molecularly identified. The fluoroprobe Spd-C2-BODIPY faithfully reports polyamine transport and accumulates almost exclusively in polyamine-sequestering vesicles (PSVs). Polyamines might thus be imported first by a plasma membrane carrier and then sequestered into pre-existing PSVs (model A), or be directly captured by polyamine receptors undergoing endocytosis (model B). Spd-C2-BODIPY uptake was unaffected in receptor-mediated endocytosis-deficient Chinese hamster ovary cell mutants. PSVs strongly colocalized with acidic vesicles of the late endocytic compartment and the trans Golgi. Virtually perfect colocalization between PSVs and acidic vesicles was found in Chinese hamster ovary cell mutants that are blocked either in the late endosome/lysosome fusion process or in the maturation of multivesicular bodies. Prior inhibition of the V-ATPase dramatically decreased total Spd-C2-BODIPY accumulation while increasing cytosolic fluorescence. Conversely, cells pre-loaded with the probe slowly released it from PSVs upon V-ATPase inhibition. The present data thus support model A, and indicate that polyamine accumulation is primarily driven by the activity of a vesicular H+:polyamine carrier.     

Detoxification of an aliphatic amine by N-acetylation: experimental and clinical studies.

Incubation of serum serpin (alpha-1-antitrypsin) with 5 mM 1,6-diaminohexane causes significant loss of heterozygotic inhibitor activity. While serpin genes have several alleles, the enzyme complex that acetylates the amine to render it suitable for excretion has primarily two phenotype populations, i.e. slow and fast N-acetylators. This study shows that diacetyl-1,6-diaminohexane does not cause in vitro loss of serpin activity, and that all regional cases (eleven in all) referred to us during one year with a diagnosis of occupational organic diisocyanate asthma were slow acetylators except one who presented a marginally fast reaction type.     

How the serpin α1-proteinase inhibitor folds

Serpins are remarkable and unique proteins in being able to spontaneously fold into a metastable conformation without the aid of a chaperone or prodomain. This metastable conformation is essential for inhibition of proteinases, so that massive serpin conformational change, driven by the favorable energetics of relaxation of the metastable conformation to the more stable one, can kinetically trap the proteinase-serpin acylenzyme intermediate. Failure to direct folding to the metastable conformation would lead to inactive, latent serpin. How serpins fold into such a metastable state is unknown. Using the ability of component peptides from the serpin α(1)PI to associate, we have now elucidated the pathway by which this serpin efficiently folds into its metastable state. In addition we have established the likely structure of the polymerogenic intermediate of the Z variant of α(1)PI.     

Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.     

Fibrillogenic C-terminal fragment of alpha-1-antitrypsin activates human monocytes via oxidative mechanisms

Production of alpha-1-antitrypsin by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases of four- to eightfold in levels of native and fragmented forms of alpha-1-antitrypsin have been detected in inflammatory loci in vivo. In this study we have extended our previous observation that the carboxyl-terminal peptide (C-36) of alpha-1-antitrypsin produced by specific proteinase cleavage, when added in its fibrillar form at concentrations of 5 microM or more to monocytes in culture, induces cytotoxic effects. Experiments with synthetic amyloid-forming peptides suggest fibril cytotoxicity to be mediated via a common oxidative stress mechanism. We undertook to determine whether C-36 fibril cytotoxicity also involves this common pathway. Monocytes stimulated with C-36 fibrils for 1 h showed significant elevation in monocyte chemoattractant protein-1 expression, induced reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, increased intracellular lipid peroxidation, altered mitochondrial membrane potential, and increased cytosolic cytochrome c and caspase-3 activity. Treatment of monocytes with C-36 fibrils after 24 h also resulted in increased cytosolic cathepsin D activity, suggesting that lysosomes may also be destabilized over longer periods of time. In contrast, native alpha-1-antitrypsin only showed concentration and time-dependent effects on chemoattractant protein-1 expression, and these appear to be independent of oxidative stress. These results indicate that the cytotoxicity of the fibrillar fragment is mediated via oxidative mechanisms and support important multiple roles for native and also for cleaved forms of alpha-1-antitrypsin in monocyte recruitment and activation during inflammatory processes such as atherosclerosis.     

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO₂ chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.