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1.
Biochim Biophys Acta ; 1798(6): 1254-62, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20346912

RESUMEN

RTA3 is an alpha-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.


Asunto(s)
Antiinfecciosos/química , Membrana Celular/química , Péptidos/química , Antiinfecciosos/farmacología , Membrana Celular/genética , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Péptidos/genética , Péptidos/farmacología , Unión Proteica , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Electricidad Estática
3.
Protein Pept Lett ; 17(11): 1334-44, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20673228

RESUMEN

Amphipathic peptides are accommodated within the diffuse gradient of polarity that characterizes the interfacial regions of phospholipid bilayer membranes. Interfacial membrane interactions are key to the diverse biological functions and activities of these peptides, which encompass a large class of antimicrobial peptides, including the helical peptides magainin, melittin, and RTA3 derived from the commensal bacterium Streptococcus mitis. For these peptides in vitro efficacy (high antimicrobial activity with minimal mammalian cell toxicity, equivalent to high potential therapeutic index; PTI), can be broadly understood in relation to the thermodynamics of interfacial binding and membrane disruption in membranes having surface charges that correspond to bacterial and mammalian cell membranes, respectively. Peptides with disrupted amphipathicity resulting from a positively charged amino acid residue on the non-polar helix face, can have greatly enhanced PTI, although a balance of amphipathicity, hydrophobicity and positive charge is required for retention of high antimicrobial activity. These observations are illustrated with recent examples from the literature, and studies on RTA3 and magainin analogues from our laboratories. Despite the identification and optimisation of peptides with very good PTI, a focus on addressing toxicity upon systemic administration and poor in vivo efficacy is likely to be required to translate growing understanding of the relationships between peptide interfacial activity and effects on cells, into novel systemic therapeutics.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Animales , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Termodinámica
4.
J Biol Chem ; 283(27): 18636-45, 2008 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-18434320

RESUMEN

We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316-12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.


Asunto(s)
Antiinfecciosos/farmacología , Proteínas Bacterianas/farmacología , Eritrocitos/metabolismo , Hemólisis/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Péptidos/farmacología , Streptococcus mitis/química , Secuencias de Aminoácidos/genética , Sustitución de Aminoácidos , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/toxicidad , Proteínas Bacterianas/síntesis química , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Eritrocitos/citología , Caballos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Mutación Missense , Péptidos/síntesis química , Péptidos/genética , Péptidos/toxicidad , Streptococcus mitis/genética , Relación Estructura-Actividad
5.
J Biol Chem ; 278(28): 25839-46, 2003 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-12738766

RESUMEN

Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells. Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease. Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function. However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear. In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385-550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers. PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric. This monomeric state was also produced at the lower PKA concentration and physiological ionic strength. Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy. The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Receptores de Superficie Celular/metabolismo , Dicroismo Circular , ADN/metabolismo , Dimerización , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Escherichia coli/patogenicidad , Guanidina/farmacología , Fosforilación , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tirosina/metabolismo , Ultracentrifugación
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