Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa

Matsuzaki, Katsumi; Mitani, Yoko; Akada, K.; Murase, Osamu; Yoneyama, Shuji; Zasloff, Michael; Miyajima, Koichiro

doi:10.1021/bi9811617

Cited by 224 publications

(318 citation statements)

References 37 publications

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“…The findings presented here, combined with previous observations that polyphemusin I promotes lipid flip-flop between membrane leaflets but does not induce significant vesicle leakage (5), rule out the torroidal pore (46) and carpet mechanisms of action (47). Differential scanning calorimetry of the pretransition and main gel to liquid-crystalline phase transition of DMPC vesicles at the indicated peptide to lipid molar ratios.…”

Section: Discussionsupporting

confidence: 79%

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,

et al. 2005

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The horseshoe crab cationic antimicrobial peptide polyphemusin I is highly active in vitro but not protective in mouse models of bacterial and LPS challenge, while a synthetic polyphemusin variant, PV5, was previously shown to be protective in vivo. In this study, we investigated the interaction of these peptides with lipid membranes in an effort to propose a mechanism of interaction. The solution structure of PV5 was determined by proton NMR in the absence and presence of dodecylphosphocholine (DPC) micelles. Like polyphemusin I, PV5 is a β-hairpin but appeared less amphipathic in solution. Upon association with DPC micelles, PV5 underwent side chain rearrangements which resulted in an increased amphipathic conformation. Using fluorescence spectroscopy, both peptides were found to have limited affinity for neutral vesicles composed of phosphatidylcholine (PC). Incorporation of 25 mol % cholesterol or phosphatidylethanolamine into PC vesicles produced little change in the partitioning of either peptide. Incorporation of 25 mol % phosphatidylglycerol (PG) into PC vesicles, a simple prokaryotic model, resulted in a large increase in the affinity for both peptides, but the partition coefficient for PV5 was almost twice that of polyphemusin I. Differential scanning calorimetry studies supported the partitioning data and demonstrated that neither peptide interacted readily with neutral PC vesicles. Both peptides showed affinity for negatively charged membranes incorporating PG. The affinity of PV5 was much greater as the pretransition peak was absent at low peptide to lipid ratios (1:400) and the reduction in enthalpy of the main transition was greater than that produced by polyphemusin I. Both peptides decreased the lamellar to inverted hexagonal phase transition temperature of PE indicating the induction of negative curvature strain. These results, combined with previous findings that polyphemusin I promotes lipid flip-flop but does not induce significant vesicle leakage, ruled out the torroidal pore and carpet mechanisms of antimicrobial action for these polyphemusins.The polyphemusins are a group of cationic peptides isolated from the American horseshoe crab, Limulus polyphemus (1), and share a great similarity to the tachyplesins from the Japanese horseshoe crab, Tachypleus tridentatus (2). These peptides are 17-18 amino acid residues in † We acknowledge funding from the Canadian Institutes of Health Research and the Applied Food and Materials Network to R.E.W.H. and NIH funding support to A.R. (AI054515). R.E.W.H. is the recipient of a Canada Research Chair, J.-P.S.P. is the recipient of a UBC postgraduate fellowship, and A.T. was supported by NIH funds (AI054515 to A.R. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript length and contain two disulfide bonds which act to constrain the peptide backbone into an antiparallel β-hairpin connected by a β-turn. To date, the solution and micelle-bound structures of tachyplesin I (3) and the solution structure of polyphemusin I (4) as we...

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Section: Discussionsupporting

confidence: 79%

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,

et al. 2005

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“…In addition, the nature of the changes to E9 DNase secondary structure is unclear, making it difficult to formulate a mechanism for its translocation. A model for the mechanism of translocation of antimicrobial cationic peptides, such as magainin-2, gramicidin S, cecropin, and melittin, has been proposed by Matsusaki (33). It has been suggested that the peptide binds initially to the membrane surface by electrostatic interactions with the polar head group of the lipids.…”

Section: Discussionmentioning

confidence: 99%

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Mosbahi

Walker

Lea

et al. 2004

Journal of Biological Chemistry

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We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476 -484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum ( max ) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in max , from 354 to 342 nm. Changes in max were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex preempts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.Unraveling the interactions and mechanisms that enable proteins to cross biological membranes is of considerable interest, as the ability to target specific exogenous enzymes to the cytosol is likely to facilitate the design and discovery of novel chemotherapeutic agents. Many protein toxins have evolved to deliver a cytotoxic domain or subunit to the cytoplasm of susceptible cells, and so they provide an invaluable tool for studying protein translocation from the extracellular environment to their cellular targets, often located in the cytoplasm (1). The transition from the water-soluble to membrane-bound state has perhaps been most intensely studied in the pore-forming colicins (2, 3). This family of bacterial toxins, like all colicins, share a common three-domain structure, with receptor-binding and translocation domains that facilitate binding to the cell surface and mediate delivery of the channel-forming cytotoxic domain to the inner membrane. Cell death occurs as a consequence of ion channel formation across the cytoplasmic membrane, inducing depolarization of the membrane. Association of the cytotoxic domain with the membrane is thought to lead to destabilization and unfolding of the protein, yielding a "molten...

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“…An explanation could be the rate at which these pores open and close. In the case of antimicrobial peptides, the time that the pore remains open is on the order of milliseconds or more [20][21][22][23][24] while in the case of the TAT peptides, we have observed that the rate is of the order of a microsecond. If the peptide has a cargo attached to it, such as a protein or a fluorescent dye, the cargo could temporarily block the pore as it translocates, reducing the leakage even further.…”

Section: Translocation Of An Arginine and Pore Formationmentioning

confidence: 99%

“…The total process takes less than a microsecond and the maximum diameter of the pore we observed was approximately 5 nm. The rate at which these pores open and close contrasts with the rate of antimicrobials peptides (order of milliseconds or more [20][21][22][23][24]) whose sequences might be specifically designed to maintain the pore open in microbial membranes for enough time to destroy the ionic concentration gradients and consequently kill the cell. This fast rate could explain why sensible leakage has not been detected [39] and why CPPs are able to translocate by forming pores without killing the host cell.…”

Section: Peptides Diffusion and Closing Of The Porementioning

confidence: 99%

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Cell Penetrating Peptides: How Do They Do It?

2007

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Cell penetrating peptides consist of short sequences of amino acids containing a large net positive charge that are able to penetrate almost any cell, carrying with them relatively large cargoes such as proteins, oligonucleotides, and drugs. During the 10 years since their discovery, the question of how they manage to translocate across the membrane has remained unanswered. The main discussion has been centered on whether they follow an energy-independent or an energy-dependent pathway. Recently, we have discovered the possibility of an energy-independent pathway that challenges fundamental concepts associated with protein-membrane interactions (Herce and Garcia, PNAS, 104: 20805 (2007) [1]). It involves the translocation of charged residues across the hydrophobic core of the membrane and the passive diffusion of these highly charged peptides across the membrane through the formation of aqueous toroidal pores. The aim of this review is to discuss the details of the mechanism and interpret some experimental results consistent with this view.

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Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa

Cited by 224 publications

References 37 publications

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Cell Penetrating Peptides: How Do They Do It?

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Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa

Cited by 224 publications

References 37 publications

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes,

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes,

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Cell Penetrating Peptides: How Do They Do It?

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Product

Resources

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Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes^,