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Biochimica Et Biophysica Acta Jul 2012Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell...
BACKGROUND
Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell death-inducing activities of four β-hairpin AMPs (gomesin, protegrin, tachyplesin and polyphemusin II) along with their linear analogs in the human erythroleukemia K562 cell line to investigate the relationship between their structure and activity.
METHODS
K562 cells were exposed to AMPs. Morphological and biochemistry alterations were evaluated using light microscopy, confocal microscopy and flow cytometry.
RESULTS
Gomesin and protegrin displayed cytotoxic properties that their linear counterparts did not. Tachyplesin and polyphemusin II and also their linear analogs induced cell death. We were able to distinguish two ways in which these AMPs induced cell death. Lower concentrations of AMPs induced controlled cell death mechanisms. Gomesin, tachyplesin and linear-tachyplesin promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation, but was also inhibited by necrostatin-1. Gomesin and protegrin induced cell death was dependent on intracellular Ca2+ mechanisms and the participation of free radicals was observed in protegrin induced cell death. Polyphemusin II and its linear analog mainly induced necrosis. Conversely, treatment with higher concentrations of AMPs primarily resulted in cell membrane disruption, but with clearly different patterns of action for each AMP tested.
CONCLUSION
Different actions by β-hairpin AMPs were observed at low concentrations and at higher concentrations despite the structure similarity.
GENERAL SIGNIFICANCE
Controlled intracellular mechanism and direct membrane disruption were clearly distinguished helping to understand the real action of AMPs in mammalian cells.
Topics: Antimicrobial Cationic Peptides; Apoptosis; Calcium; Caspases; Cell Membrane; Humans; K562 Cells; Necrosis; Reactive Oxygen Species
PubMed: 22425533
DOI: 10.1016/j.bbagen.2012.02.015 -
Chemotherapy Research and Practice 2011Superantigens are proteins comprising a group of molecules produced by various microorganisms. They are involved in pathogenesis of several human diseases. The aim of...
Activity of Antimicrobial Peptides and Conventional Antibiotics against Superantigen Positive Staphylococcus aureus Isolated from the Patients with Neoplastic and Inflammatory Erythrodermia.
Superantigens are proteins comprising a group of molecules produced by various microorganisms. They are involved in pathogenesis of several human diseases. The aim of the study was the comparison of susceptibility to antibiotics and antimicrobial peptides (AMPs) of Staphylococcus aureus (SA) strains producing staphylococcal enterotoxins SEA, SEB, SEC, SED, and TSST-1 and nonproducing ones. In the group of the total 28 of the patients with erythrodermia the presence of SA was confirmed in 24 cases. The total of 14 strains of SA excreted enterotoxins SEA, SEC, SED, and TSST-1. We did not observe that strains producing mentioned superantigens were less susceptible to AMPs (aurein 1.2, citropin 1.1, lipopeptide, protegrin 1, tachyplesin 3, temporin A, and uperin 3.6). The opposite situation was observed in conventional antibiotics. SA strains excreting tested superantigens had higher MICs and MBCs than nonproducing ones. The interesting finding considering the high efficacy of AMPs, against all examined strains of SA, makes them attractive candidates for therapeutic implication.
PubMed: 22312551
DOI: 10.1155/2011/270932 -
Biochimica Et Biophysica Acta Feb 2012It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the...
It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.
Topics: Antimicrobial Cationic Peptides; Cell Membrane; Dimyristoylphosphatidylcholine; Membrane Lipids; Molecular Sequence Data; Molecular Structure; Protein Structure, Secondary
PubMed: 22100601
DOI: 10.1016/j.bbamem.2011.11.002 -
Molecular Pharmaceutics Apr 2012More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous... (Review)
Review
More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape β-strand-turn-β-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.
Topics: Amyloid; Amyloid beta-Peptides; Animals; Anti-Infective Agents; Electrophysiology; Humans
PubMed: 22081976
DOI: 10.1021/mp200419b -
The Journal of Physical Chemistry. B Dec 2011Molecular dynamics (MD) simulations are used to study the pathway for the insertion of the cationic antimicrobial peptide protegrin-1 (PG1) into mixed anionic lipid...
Molecular dynamics (MD) simulations are used to study the pathway for the insertion of the cationic antimicrobial peptide protegrin-1 (PG1) into mixed anionic lipid bilayers composed of palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG/POPE). We calculate the potential of mean force (PMF) during the transfer of the peptide from the bulk aqueous phase to the transmembrane (TM) configuration using the adaptive biasing force (ABF) method. We find that the PMF has two energy minima separated by an energy barrier. One minimum corresponds to the fully transmembrane inserted state, with a free energy of -20.1 kcal/mol. The second PMF minimum, which corresponds to adsorption to the membrane surface, has a value of -2.5 kcal/mol. The PMF also shows the existence of a free energy barrier of +6.3 kcal/mol for the insertion process. Using the Kramers theory Langevin equation and the Grote-Hynes theory generalized Langevin equation, we calculated the transmission coefficient for PG1 diffusion through the potential barrier. We focus on the use of the PMF and the time correlation function of the fluctuation of the instantaneous force to calculate the rate constants for insertion/deinsertion of PG1 from the mixed POPG/POPE membrane. The influence of the activation free energy barrier on the dynamics of the insertion and permeation of peptides through the membrane are discussed.
Topics: Antimicrobial Cationic Peptides; Lipid Bilayers; Molecular Dynamics Simulation; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics
PubMed: 22044268
DOI: 10.1021/jp205153y -
Biophysical Journal Apr 2011Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a...
Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.
Topics: Adsorption; Aluminum Silicates; Amyloid; Antimicrobial Cationic Peptides; Computer Simulation; Kinetics; Lipid Bilayers; Microscopy, Atomic Force; Protein Structure, Secondary; Time Factors
PubMed: 21463591
DOI: 10.1016/j.bpj.2011.01.072 -
Biophysical Journal Apr 2011
Topics: Alzheimer Disease; Amyloid; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Cell Membrane Permeability; Fungi; Humans; Protein Structure, Secondary
PubMed: 21463571
DOI: 10.1016/j.bpj.2011.02.023 -
Protein Science : a Publication of the... Apr 2011Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid...
Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein-lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides.
Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Cations; Cell-Penetrating Peptides; DNA-Binding Proteins; Humans; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Peptides; Peptides, Cyclic; Potassium Channels, Voltage-Gated; Protein Conformation; alpha-Defensins
PubMed: 21344534
DOI: 10.1002/pro.600 -
Biochemistry Mar 2011The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic...
Structures of β-hairpin antimicrobial protegrin peptides in lipopolysaccharide membranes: mechanism of gram selectivity obtained from solid-state nuclear magnetic resonance.
The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.
Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Biomimetics; Cell Membrane; Gram-Negative Bacteria; Gram-Positive Bacteria; Guanidine; Lipopolysaccharides; Microbial Sensitivity Tests; Models, Molecular; Molecular Sequence Data; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Species Specificity
PubMed: 21302955
DOI: 10.1021/bi101975v -
Biochemistry Nov 2010Defensins are cationic and disulfide-bonded host defense proteins of many animals that target microbial cell membranes. Elucidating the three-dimensional structure,...
Defensins are cationic and disulfide-bonded host defense proteins of many animals that target microbial cell membranes. Elucidating the three-dimensional structure, dynamics, and topology of these proteins in phospholipid bilayers is important for understanding their mechanisms of action. Using solid-state nuclear magnetic resonance spectroscopy, we have now determined the conformation, dynamics, oligomeric state, and topology of a human α-defensin, HNP-1, in DMPC/DMPG bilayers. Two-dimensional correlation spectra show that membrane-bound HNP-1 exhibits a conformation similar to that of the water-soluble state, except for the turn connecting strands β2 and β3, whose side chains exhibit immobilization and conformational perturbation upon membrane binding. At high protein/lipid ratios, rapid (1)H spin diffusion from the lipid chains to the protein was observed, indicating that HNP-1 was well inserted into the hydrocarbon core of the bilayer. Arg Cζ-lipid (31)P distances indicate that only one of the four Arg residues forms tight hydrogen-bonded guanidinium-phosphate complexes. The protein is predominantly dimerized at high protein/lipid molar ratios, as shown by (19)F spin diffusion experiments. The presence of a small fraction of monomers and the shallower insertion at lower protein concentrations suggest that HNP-1 adopts concentration-dependent oligomerization and membrane-bound structure. These data strongly support a "dimer pore" topology of HNP-1 in which the polar top of the dimer lines an aqueous pore while the hydrophobic bottom faces the lipid chains. In this structure, R25 lies closest to the membrane surface among the four Arg residues. The pore does not have a high degree of lipid disorder, in contrast to the toroidal pores formed by protegrin-1, a two-stranded β-hairpin antimicrobial peptide. These results provide the first glimpse into the membrane-bound structure and mechanism of action of human α-defensins.
Topics: Diffusion; Dimerization; Humans; Hydrogen Bonding; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation; Water; alpha-Defensins
PubMed: 20961099
DOI: 10.1021/bi101512j