Seung-Hwan Jang

A Leu-Lys-rich antimicrobial peptide: activity and mechanism.

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.

Fungicidal effect of indolicidin and its interaction with phospholipid membranes.

The fungicidal effect and mechanism of a tryptophan-rich 13-mer peptide, indolicidin derived from granules of bovine neutrophils, were investigated. Indolicidin displayed a strong fungicidal activity against various fungi. In order to understand the fungicidal mechanism(s) of indolicidin, we examined the interaction of indolicidin with the pathogenic fungus Trichosporon beigelii. Fluorescence confocal microscopy and flow cytometry analysis revealed that indolicidin acted rapidly on the plasma membrane of the fungal cells in an energy-independent manner. This interaction is also dependent on the ionic environment. Furthermore, indolicidin caused significant morphological changes when tested for the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w). The results suggest that indolicidin may exert its fungicidal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers, in a salt-dependent and energy-independent manner.

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella typhimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the α-helical structure of these peptides plays an important role in their antibiotic effect but that the α-helical property is not connected with the enhanced antibiotic activity.

Analysis of the Thermostability Determinants of Hyperthermophilic Esterase EstE1 Based on Its Predicted Three-Dimensional Structure

ABSTRACT The three-dimensional (3D) structure of the hyperthermophilic esterase EstE1 was constructed by homology modeling using Archaeoglobus fulgidus esterase as a reference, and the thermostability-structure relationship was analyzed. Our results verified the predicted 3D structure of EstE1 and identified the ion pair networks and hydrophobic interactions that are critical determinants for the thermostability of EstE1.

Antinematodal activity and the mechanism of the antimicrobial peptide, HP (2-20), against Caenorhabditis elegans

The peptide HP (2-20), derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1), has a nematicidal activity against eggs and worms of Caenorhabditis elegans. Eggs treated with HP (2-20) (69%) has a higher fluorescence intensity with propidium iodide staining, which was similar to that of melittin (82%) but higher than untreated cells (5.7%). Confocal microscopy showed that the peptides were located in the shell of the eggs and the inner and outer surfaces of the worms. HP (2-20) therefore may exert its antinematodal activity by disrupting the structure of the eggs shell and the cell membrane via pore formation or by direct interaction with the lipid bilayers in a detergent-like manner.

A New Approach to Find Orthologous Proteins Using Sequence and Protein-Protein Interaction Similarity

Developed proteome-scale ortholog and paralog prediction methods are mainly based on sequence similarity. However, it is known that even the closest BLAST hit often does not mean the closest neighbor. For this reason, we added conserved interaction information to find orthologs. We propose a genome-scale, automated ortholog prediction method, named OrthoInterBlast. The method is based on both sequence and interaction similarity. When we applied this method to fly and yeast, 17% of the ortholog candidates were different compared with the results of Inparanoid. By adding protein-protein interaction information, proteins that have low sequence similarity still can be selected as orthologs, which can not be easily detected by sequence homology alone.