Su A Jang

Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide

Buforin IIb is a novel cell-penetrating anticancer peptide derived from histone H2A. Here we analyzed the anticancer activity and cancer cell-killing mechanism of buforin IIb. Buforin IIb displayed selective cytotoxicity against 62 cancer cell lines by specifically targeting cancer cells through interaction with cell surface gangliosides. It traversed cancer cell membranes without damaging them and accumulated primarily in the nuclei. Once inside the cells, buforin IIb induced mitochondria-dependent apoptosis. In vivo analysis revealed that buforin IIb displayed significant tumor suppression activity in mice with tumor xenograft. Overall, these results suggest that buforin IIb constitutes a novel therapeutic agent for the treatment of cancers.

Engineering butanol-tolerance in escherichia coli with artificial transcription factor libraries.

Escherichia coli has been explored as a host for butanol production because of its many advantages such as a fast growth and easy genetic manipulation. Butanol toxicity, however, is a major concern in the biobutanol production with E. coli. In particular, E. coli growth is severely inhibited by butanol, being almost completely stopped by 1% (vol/vol) butanol. Here we developed a new method to increase the butanol‐tolerance of E. coli with artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA‐binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we selected a butanol‐tolerant E. coli which can tolerate up to 1.5% (vol/vol) butanol, with a concomitant increase in heat resistance. We also identified genes of E. coli that are associated with the butanol‐tolerance. These results show that E. coli can be engineered as a promising host for high‐yield butanol production. Biotechnol. Bioeng. 2011; 108:742–749.

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.

Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

ABSTRACT We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of Escherichia coli culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.