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Il-Seon Park

Publications and source records attributed to Il-Seon Park.

14 recordsLinked to original sources
PubMed2006

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide.

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.

Amino Acid Sequence↗
PubMed2006

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1.

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.

Amino Acid Sequence↗
PubMed2006

Mechanism of antibacterial action of a synthetic peptide with an Ala-peptoid residue based on the scorpion-derived antimicrobial peptide IsCT.

A novel bacterial cell-selective antimicrobial peptide, IsCT-P (ILKKIWKPIKKLF-NH(2)), was designed based on the scorpion-derived alpha-helical antimicrobial peptide, IsCT. Here, we investigated the effect of substituting Pro(8) of IsCT-P with the Ala-peptoid residue (N-methylglycine) on the peptide's structure and mechanism of action. Circular dichroism analysis revealed that the modified peptide, IsCT-a, has a much lower alpha-helicity than IsCT-P in membrane mimicking conditions, suggesting the peptoid residue provides much more structural flexibility than the proline residue. IsCT-a was also much less effective than IsCT-P at causing leakage of fluorescent dye entrapped within negatively charged vesicles and at dissipating the membrane potential of Staphylococcus aureus. Collectively, our results suggest that the antibacterial action of IsCT-a is due to the inhibition of intracellular targets rather than the disruption and depolarization of bacterial cell membranes.

Amino Acid Sequence↗
PubMed2006

Comparative study of apoptosis induced by H(2)O(2) and NO: limitation of apoptosis induced by NO due to slower recovery of activity of NO-modified caspase.

Both H(2)O(2) and NO can act as apoptogens, triggering apoptosis in many cells. They are also well known inhibitors of caspases, essential enzymes in apoptosis. The differences between these two agents as apoptosis inducers and how caspases mediate apoptosis with these inhibitory agents is still unclear. Consistent with the previous reports, these two agents induced apoptosis accompanied by caspase activation with limitation of all apoptotic events for NO. It was found that NO-modified caspase-3 showed a slower recovery of its activity in the presence of the reducing agents compared to that of H(2)O(2) modification. This is one possible cause of the limited apoptosis in the case of NO.

Apoptosis↗
PubMed2006

Improvement of bacterial cell selectivity of melittin by a single Trp mutation with a peptoid residue.

To design melittin (ME) analogues that are not cytotoxic against mammalian cells but which possessing potent antimicrobial activity, we synthesized a ME analogue (ME-w) in which the Trp-19 residue of ME was replaced by a Trp-peptoid residue (Nhtrp). ME-w exhibited similar antimicrobial activity compared to ME against the tested six bacteria and C. albicans. However, it was much less cytotoxic against the hRBCs and HeLa and NIH-3T3 cells than ME. Tryptophan fluorescence and CD spectra revealed that the Trp-19 --> Nhtrp substitution in ME contributed to a much lower helical assembly in an aqueous environment and structural flexibility and exterior localization to zwitterionic membrane which modulates its selectivity toward bacterial cells.

Amino Acid Sequence↗
PubMed2005

Kinetic comparison of procaspase-3 and caspase-3.

Caspases, the key enzymes in apoptosis, are synthesized as proenzymes and converted into active form by proteolytic cleavage. The residues on active site reorganize during the activation process as shown in the comparative studies of crystallographic structures of procaspase-7 and its mature form. On the other hand, the proenzyme itself has some activity. Aiming to characterize the activation process, the comparative kinetic study for the pro- and mature caspase-3 was performed. In 1/K(M) versus pH study, a residue with pKa of 6.89+/-0.13 was detected only in caspase-3. While Vmax versus pH kinetic results were consistent with the existence of a residue with pKa of 6.21+/-0.06 in procaspase-3 mutant (D9A/D28A/D175A) but not in caspase-3. In the inactivation assays with diethylpyrocarbonate, a residue (pKa, 6.61+/-0.05) could be determined only for caspase-3 whereas with iodoacetamide a residue with pKa value (6.01+/-0.05) could be assigned only for procaspase-3. Considering that those residues could be protected by caspase-3-specific inhibitor from the inactivation, the modifiers are histidine- and cysteine-specific, respectively, and the involvement of these residues in the characteristic catalytic dyad of caspases, the results indicate that the pKa values of the catalytic histidine and cysteine residues are changed during the activation process.

Binding Sites↗
PubMed2005

Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues.

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.

Amino Acid Sequence↗
PubMed2005

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide.

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.

Antimicrobial Cationic Peptides↗
PubMed2005

Salt is necessary for nucleosomal DNA fragmentation induced by caspase.

For nucleosomal DNA fragmentation, one of the hallmarks of apoptosis, activated caspase, an apoptosis specific cysteine protease, is required to cleave ICAD/DFF45 that releases its complexed DNase, CAD/DFF40. The protein complex is located predominantly in the nuclei. Inconsistently, caspase alone cannot induce DNA fragmentation in the isolated nuclei without the addition of a cell extract or purified CAD/DFF40. In this study, however, it is demonstrated that under selected conditions with 50-75 mM: KCl or NaCl, caspase-3 and-7 can induce DNA fragmentation without the additional factor(s).

Animals↗
PubMed2005

Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis.

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.

Cations↗
PubMed2004

Rearrangement of tryptophan residues in caspase-3 active site upon activation.

Caspase-3, one of the major apoptotic proteins, is a cysteine protease and exists as an inactive zymogen in healthy cells. In this study, the dynamic nature of the rearrangements of two tryptophan residues (Trp 206 and Trp 214) in the active sites of caspase-3 during the activation was analyzed by measuring the fluorescence lifetimes. Significant changes in the lifetime occurred upon activation by the specific cleavage. In addition, two mutant proteins that have only one tryptophan residue also showed the similar changes. These data indicate that the activation of caspase-3 resulted in the reorganization of both tryptophan residues.

Binding Sites↗
PubMed2002

Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula.

Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.

Amino Acid Sequence↗
PubMed2002

Biochemical characterization of apoptotic cleavage of KH-type splicing regulatory protein (KSRP)/far upstream element-binding protein 2 (FBP2).

Caspases, Asp-specific cysteine protease, cleave proteins upon apoptosis. To identify and characterize new caspase substrate in the nucleus, the proteome of the rat liver extracts was analyzed after the treatment with caspases. One of the identified proteins was KSRP/FBP2 that is preferentially cleaved by caspase-3 and 7 at two sites after Asp102 and Asp183. The second site was cleaved only in the protein produced in cells, but not in in vitro translated protein. These results indicate that more than the primary sequence may be important for the recognition by caspases.

Animals↗