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Biophysical Journal Jan 2014Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed...
Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.
Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Lipid Bilayers; Molecular Dynamics Simulation; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols
PubMed: 24411247
DOI: 10.1016/j.bpj.2013.11.4490 -
Biophysical Journal Nov 2013Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to...
Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.
Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Bacteria; Carrier Proteins; Cell Membrane; Cell-Penetrating Peptides; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols; Temperature; tat Gene Products, Human Immunodeficiency Virus
PubMed: 24268145
DOI: 10.1016/j.bpj.2013.08.020 -
Acta Poloniae Pharmaceutica 2013Antimicrobial peptides (AMPs) are an essential part of the innate immunity of the skin and mucosal surfaces. They have a broad spectrum of antimicrobial activity:...
Antimicrobial peptides (AMPs) are an essential part of the innate immunity of the skin and mucosal surfaces. They have a broad spectrum of antimicrobial activity: antibacterial, antifungal, antiviral as well as antiprotozoal. Numerous studies using AMPs as potential agents against different microbes has been performed during the last two decades. Here we investigated antistaphylococcal activity and safety of following AMPs: camel, citropin, protegrin, temporin A and lipopeptide Palm-KK-NH2. The susceptibility of the strains of Staphylococcus aureus isolated from the patients with erythrodermia to conventional antibiotics and AMPs was determined by the broth dilution method. The cytotoxicity assay was performed on HaCaT keratinocytes. Tested peptides turned out to be very effective against all clinical isolates, including strains resistant to conventional antibiotics. The majority of the examined peptides as well as conventional antimicrobials do not exert any toxic effect on HaCaT cells in minimal inhibitory concentration. Peptides are very promising agents for the topical treatment of staphylococcal skin infections. The most promising seem to be citropin 1.1 and temporin A, as they were toxic only in two highest concentration (50 and 100 microg/mL), with relatively low MIC values.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Line; Cell Proliferation; Cell Survival; Humans; Keratinocytes; Microbial Sensitivity Tests; Proteins; Staphylococcal Infections; Staphylococcus aureus
PubMed: 24147357
DOI: No ID Found -
PloS One 2013Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly...
Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2), pBD3, epididymis protein 2 splicing variant C (pEP2C), and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.
Topics: Animals; Butyrates; Cell Line; Enterocytes; Fatty Acids, Volatile; Gene Expression Regulation; Macrophages; Monocytes; Sus scrofa; beta-Defensins
PubMed: 24023657
DOI: 10.1371/journal.pone.0072922 -
Biophysical Journal Feb 2013Protegrin is an antimicrobial peptide with a β-hairpin structure stabilized by a pair of disulfide bonds. It has been extensively studied by solid-state NMR and...
Protegrin is an antimicrobial peptide with a β-hairpin structure stabilized by a pair of disulfide bonds. It has been extensively studied by solid-state NMR and computational methods. Here we use implicit membrane models to examine the binding of monomers on the surface and in the interior of the membrane, the energetics of dimerization, the binding to membrane pores, and the stability of different membrane barrel structures in pores. Our results challenge a number of conclusions based on previous experimental and theoretical work. The burial of monomers into the membrane interior is found to be unfavorable for any membrane thickness. Because of its imperfect amphipathicity, protegrin binds weakly, at most, on the surface of zwitterionic membranes. However, it binds more favorably onto toroidal pores. Anionic charge on the membrane facilitates the binding due to electrostatic interactions. Solid-state NMR results have suggested a parallel NCCN association of monomers in dimers and association of dimers to form octameric or decameric β-barrels. We find that this structure is not energetically plausible for binding to bilayers, because in this configuration the hydrophobic sides of two monomers point in opposite directions. In contrast, the antiparallel NCCN and especially the parallel NCNC octamers are stable and exhibit a favorable binding energy to the pore. The results of 100-ns simulations in explicit bilayers corroborate the higher stability of the parallel NCNC barrel compared with the parallel NCCN barrel. The ability to form pores in zwitterionic membranes provides a rationalization for the peptide's cytotoxicity. The discrepancies between our results and experiment are discussed, and new experiments are proposed to resolve them and to test the validity of the models.
Topics: Amino Acid Sequence; Anions; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Membrane; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Lipids; Molecular Dynamics Simulation; Molecular Sequence Data; Protein Binding; Protein Multimerization; Protein Subunits; Static Electricity
PubMed: 23442914
DOI: 10.1016/j.bpj.2012.12.038 -
Biochemistry Mar 2013Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large...
Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.
Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cathepsin L; Crystallography, X-Ray; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; Models, Molecular; Protein Folding; Protein Structure, Tertiary; Proteins; Swine; Cathelicidins
PubMed: 23406372
DOI: 10.1021/bi301008r -
Journal of Chemical Theory and... Jan 2013Amyloid-β (Aβ) oligomers destabilize cellular ionic homeostasis, mediating Alzheimer's disease (AD). It is still unclear whether the mechanism (i) is mediated by cell...
Amyloid-β (Aβ) oligomers destabilize cellular ionic homeostasis, mediating Alzheimer's disease (AD). It is still unclear whether the mechanism (i) is mediated by cell surface receptors; (ii) is direct, with Aβ oligomers interacting with membrane lipids; or (iii) both mechanisms take place. Recent studies indicate that Aβ oligomers may act by either of the last two. Little is known about the oligomers' structures and how they spontaneously insert into the membrane. Using explicit solvent molecular dynamics (MD) simulations, we show that fibril-like Aβ(17-42) (p3) oligomer is capable of penetrating the membrane. Insertion is similar to that observed for protegrin-1 (PG-1), a cytolytic β-sheet-rich antimicrobial peptide (AMP). Both Aβ and PG-1 favor the amphipathic interface of the lipid bilayer in the early stage of interaction with the membrane. U-shaped Aβ oligomers are observed in solution and in the membrane, suggesting that the preformed seeds can be shared by amyloid fibrils in the growth phase and membrane toxicity. Here we provide sequential events in possible Aβ oligomer membrane-insertion pathways. We speculate that for the U-shaped motif, a trimer is the minimal oligomer size to insert effectively. We propose that monomers and dimers may insert in (apparently on-pathway) aggregation-intermediate β-hairpin state, and may (or may not) convert to a U-shape in the bilayer. Together with earlier observations, our results point to a non-specific, broadly heterogeneous landscape of membrane-inserting oligomer conformations, pathways, and membrane-mediated toxicity of β-rich oligomers.
PubMed: 23316126
DOI: 10.1021/ct300916f -
International Journal of Molecular... 2012Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their... (Review)
Review
Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their unique mechanism of action, they have been touted as a potential source for novel antibiotic drugs. We present a summary of computational investigations in our lab aimed at understanding this unique mechanism of action, in particular the development of models that provide a quantitative connection between molecular-level biophysical phenomena and relevant biological effects. Our work is focused on protegrins, a potent class of AMPs that attack bacteria by associating with the bacterial membrane and forming transmembrane pores that facilitate the unrestricted transport of ions. Using fully atomistic molecular dynamics simulations, we have computed the thermodynamics of peptide-membrane association and insertion, as well as peptide aggregation. We also present a multi-scale analysis of the ion transport properties of protegrin pores, ranging from atomistic molecular dynamics simulations to mesoscale continuum models of single-pore electrodiffusion to models of transient ion transport from bacterial cells. Overall, this work provides a quantitative mechanistic description of the mechanism of action of protegrin antimicrobial peptides across multiple length and time scales.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane; Gram-Negative Bacteria; Ion Channels; Ion Transport; Microbial Sensitivity Tests; Models, Molecular; Models, Theoretical; Molecular Dynamics Simulation; Thermodynamics
PubMed: 23109834
DOI: 10.3390/ijms130911000 -
Journal of Biomedicine & Biotechnology 2012Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards...
Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC(50) of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P < 0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P < 0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.
Topics: Animals; Antimicrobial Cationic Peptides; Cell Line; Dengue Virus; Enzyme Inhibitors; Kidney; Macaca mulatta; Serine Endopeptidases; Viral Nonstructural Proteins; Virus Replication
PubMed: 23093838
DOI: 10.1155/2012/251482 -
Microbiology and Immunology Aug 2012Early defence mechanisms of innate immunity respond rapidly to infection against HIV-1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive... (Review)
Review
Early defence mechanisms of innate immunity respond rapidly to infection against HIV-1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive immune responses against persistent HIV infection. Recent research has highlighted the intrinsic roles of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G, tripartite motif-containing protein 5, tetherin, sterile α-motif and histidine/aspartic acid domain-containing protein 1 in restricting HIV-1 replication. Likewise, certain endogenously secreted antimicrobial peptides, namely α/β/θ-defensins, lactoferrins, secretory leukocyte protease inhibitor, trappin-2/elafin and macrophage inflammatory protein-3α are reportedly protective. Whilst certain factors directly inhibit HIV, others can be permissive. Interferon-λ3 exerts an anti-HIV function by activating Janus kinase-signal transducer and activator of transcription-mediated innate responses. Morphine has been found to impair intracellular innate immunity, contributing to HIV establishment in macrophages. Interestingly, protegrin-1 could be used therapeutically to inhibit early HIV-1 establishment. Moreover, chloroquine inhibits plasmacytoid dendritic cell activation and improves effective T-cell responses. This minireview summarizes the recently identified targets for innate immunity-mediated therapies and outlines the challenges that lie ahead in improving treatment of HIV infection.
Topics: HIV Infections; HIV-1; Humans; Immunity, Innate; Immunologic Factors; Immunotherapy
PubMed: 22900503
DOI: 10.1111/j.1348-0421.2012.00485.x