Janet Hammer

Antimicrobial Activities and Structures of Two Linear Cationic Peptide Families with Various Amphipathic β-Sheet and α-Helical Potentials

ABSTRACT Many naturally occurring antimicrobial peptides comprise cationic linear sequences with the potential to adopt an amphipathic α-helical conformation. We designed a linear 18-residue peptide that adopted an amphipathic β-sheet structure when it was bound to lipids. In comparison to a 21-residue amphipathic α-helical peptide of equal charge and hydrophobicity, this peptide possessed more similar antimicrobial activity and greater selectivity in binding to and inducing leakage in vesicles composed of bacterial membrane lipids than vesicles composed of mammalian membrane lipids (J. Blazyk, R. Weigand, J. Klein, J. Hammer, R. M. Epand, R. F. Epand, W. L. Maloy, and U. P. Kari, J. Biol. Chem. 276:27899-27906, 2001). Here, we compare two systematically designed families of linear cationic peptides to evaluate the importance of amphipathicity for determination of antimicrobial activity. Each peptide contains six lysine residues and is amidated at the carboxyl terminus. The first family consists of five peptides with various capacities to form amphipathic β-sheet structures. The second family consists of six peptides with various potentials to form amphipathic α helices. Only those peptides that can form a highly amphipathic structure (either a β sheet or an α helix) possessed significant antimicrobial activities. Striking differences in the abilities to bind to and induce leakage in membranes and lipid vesicles were observed for the two families. Overall, the amphipathic β-sheet peptides are less lytic than their amphipathic α-helical counterparts, particularly toward membranes containing phosphatidylcholine, a lipid commonly found in mammalian plasma membranes. Thus, it appears that antimicrobial peptides that can form an amphipathic β-sheet conformation may offer a selective advantage in targeting bacterial cells.

Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles

1. In rat skeletal muscles (longissimus dorsi and quadriceps femoris), carnosine and anserine levels decreased 35-50% during senescence, and were 35-45% lower in hypertensive rats compared to normotensive levels. 2. In rat left ventricular cardiac muscle, although no free carnosine and anserine were detected, the total level of histidine dipeptides declined 22% during senescence and in hypertensive animals decreased 35% compared to normotensive levels. 3. The significance of these changes in relation to the possible antioxidant roles of histidine dipeptides in muscle is discussed.

Calpain and calpastatin levels in dystrophic hamster skeletal muscles.

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.

Effects of L-1-methyl-histidine and the muscle dipeptides carnosine and anserine on the activities of muscle calpains.

1. Carnosine, anserine and L-1-methyl-histidine activated muscle calpain II assayed at 2.5 mM Ca2+. 2. At 5 microM Ca2+, none of these compounds activated calpain II sufficiently to bring its activity up to the level measured at 2.5 mM Ca2+. 3. Carnosine increased, whereas both anserine and L-1-methyl-histidine decreased the inhibitory effect of calpastatin on calpain II. 4. These results suggest that although the compounds are not potent activators of calpain II, the ratio of the dipeptides in muscle may have an effect on calpain II-calpastatin interaction.

Cadiac and skeletal muscle enzymes levels in hypertensive and ageing rats

Abstract 1. 1. The activities of glycolytic, fatty acid oxidation and citric acid cycle enzymes were measured in hypertensive and ageing rat cardiac and skeletal muscles. 2. 2. Lactate dehydrogenase and β-hydroxyacyl-CoA dehydrogenase were significally decreased in hypertensive, but not senescent, cardiac muscle. 3. 3. Total phosphorylase activity was significantly increased in senescent, but not hypertensive, cardiac muscle. 4. 4. In ageing rat cardiac and skeletal muscles, calpian II titers increased significantly with age, but in normotensive and hypertensive muscles, the titers showed no significant difference.

Complementary Effects of Host Defense Peptides Piscidin 1 and Piscidin 3 on DNA and Lipid Membranes: Biophysical Insights into Contrasting Biological Activities

Piscidins were the first antimicrobial peptides discovered in the mast cells of vertebrates. While two family members, piscidin 1 (p1) and piscidin 3 (p3), have highly similar sequences and α-helical structures when bound to model membranes, p1 generally exhibits stronger antimicrobial and hemolytic activity than p3 for reasons that remain elusive. In this study, we combine activity assays and biophysical methods to investigate the mechanisms underlying the cellular function and differing biological potencies of these peptides, and report findings spanning three major facets. First, added to Gram-positive (Bacillus megaterium) and Gram-negative (Escherichia coli) bacteria at sublethal concentrations and imaged by confocal microscopy, both p1 and p3 translocate across cell membranes and colocalize with nucleoids. In E. coli, translocation is accompanied by nonlethal permeabilization that features more pronounced leakage for p1. Second, p1 is also more disruptive than p3 to bacterial model membranes, as quantified by a dye-leakage assay and (2)H solid-state NMR-monitored lipid acyl chain order parameters. Oriented CD studies in the same bilayers show that, beyond a critical peptide concentration, both peptides transition from a surface-bound state to a tilted orientation. Third, gel retardation experiments and CD-monitored titrations on isolated DNA demonstrate that both peptides bind DNA but p3 has stronger condensing effects. Notably, solid-state NMR reveals that the peptides are α-helical when bound to DNA. Overall, these studies identify two polyreactive piscidin isoforms that bind phosphate-containing targets in a poised amphipathic α-helical conformation, disrupt bacterial membranes, and access the intracellular constituents of target cells. Remarkably, the two isoforms have complementary effects; p1 is more membrane active, while p3 has stronger DNA-condensing effects. Subtle differences in their physicochemical properties are highlighted to help explain their contrasting activities.

Effects of calpain on antioxidant enzyme activities.

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.

Fine-Tuning the Activity of Linear Amphipathic Beta-Sheet Antimicrobial Peptides

It is relatively simple to design highly amphipathic linear cationic beta-sheet peptides containing 10-to-11 amino acids that possess potent antimicrobial activity. Usually, however, these peptides also are quite hemolytic, so that there is insufficient selectivity between bacterial and human cells. Peptides with little or no hemolytic (or other toxic) activity toward host cells at 100 or more times the minimum inhibitory concentrations toward bacterial cells might be potential candidates for clinical use as antimicrobials. We have used two strategies to separately attenuate lytic activity toward host cells while maintaining potent antimicrobial activity. Both strategies involve introducing a structural perturbation in the amphipathic beta sheet. First, a hydrophobic amino acid residue can be substituted by proline. Depending upon the location of the substitution within the peptide, it is possible to nearly eliminate hemolytic activity while retaining potent antimicrobial activity. A similar outcome can be achieved by replacing a hydrophobic amino acid residue with a D-amino acid. Here again, the location of the substitution within the peptide is critical for the desired balance of activities. We show here 10- and 11-residue peptides consisting of alternating lysine and leucine in which a single leucine has been replaced by either proline or a D-amino acid. The effects of these substitutions on antimicrobial and hemolytic activities, secondary structure, and ability to induce leakage in lipid vesicles and bacterial cells are compared. The most promising peptides will be tested in vivo to determine their suitability as either topical or systemic antimicrobial agents.

Relationship Between Amphipathic Secondary Structure and Activity in Model Linear Cationic Antimicrobial Peptides

Cationic peptides are a common component of many host defense systems [1]. Upon binding to the plasma membrane of target bacteria, many of these peptides can adopt a conformation that exhibits amphipathic character. The accumulation of peptides at the membrane surface eventually results in a breakdown of the permeability barrier in the target organism, resulting in cell death. Naturally occurring cationic antimicrobial peptides usually fall into one of two classes. Linear peptides such as magainins, PGLa and cecropins, adopt α-helical structure, while cysteine-containing peptides such as defensins (constrained by intramolecular disulfide bonds) form β-sheet conformation in order to maximize amphipathicity. Recently we designed an 18-residue linear peptide, (KIGAKI)3-NH2, with no amphipathic propensity as an α-helix, but a highly amphipathic β-sheet structure [2]. Although this peptide possesses similar antimicrobial activity to that of linear a-helical peptides of comparable size, charge and hydrophobicity, it appears to be significantly more selective for bacterial membranes as compared to mammalian membranes. In this study, we examined the relationship between amphipathicity, secondary structure, antimicrobial activity, and lipid selectivity among a representative group of model peptides.