temporin and Inflammation

Temporin-1CEa, which is isolated from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria and antitumor activity. LK2(6) and LK2(6)A(L) are the analogs of temporin-1CEa obtained by replacing amino acids and displayed an improved anticancer activity. In the present study, the anti-inflammatory activity and mechanism of action of temporin-1CEa and its analogs LK2(6) and LK2(6)A(L) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages were investigated. The results showed that temporin-1CEa and its analogs decreased the production of the cytokines tumor necrosis factor-α and interleukin-6 by inhibiting the protein expression of nuclear factor-κB and mitogen-activated protein kinase and the MyD88-dependent signaling pathway. Isothermal titration calorimetry studies revealed that temporin-1CEa, LK2(6) and LK2(6)A(L) exhibited binding affinities to LPS, an important inflammatory inducer, with Kd values of 0.1, 0.03 and 0.06 μM, respectively. Circular dichroism and zeta potential experiments showed that temporin-1CEa and its analogs interacted with LPS by electrostatic binding between the positively charged peptides and negatively charged LPS, resulting in the neutralization of LPS toxicity.

Topics: Animals; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Cell Line; Inflammation; Lipopolysaccharides; Macrophages; Mice; Myeloid Differentiation Factor 88; Pore Forming Cytotoxic Proteins; Ranidae; RAW 264.7 Cells; Signal Transduction; Skin

Temporin L (TempL) is a 13-residue frog antimicrobial peptide that shows moderate bactericidal activity and antiendotoxin properties in macrophages. We envisioned that, due to its very hydrophobic nature, the peptide might fail to show its desired biological properties. It was predicted by employing the available algorithms that the replacement of a glutamine by lysine at position 3 could appreciably reduce its aggregation propensity in an aqueous environment. In order to investigate the structural, functional, and biological consequences of replacement of glutamine by lysine at its third position, TempL and the corresponding analog, Q3K-TempL, was synthesized and characterized. Introduction of the lysine residue significantly promoted the self-assembly and oligomeric state of TempL in lipopolysaccharide (LPS). Q3K-TempL exhibited augmented binding to LPS and also dissociated LPS aggregates with greater efficacy than TempL. Further, Q3K-TempL inhibited the LPS-induced proinflammatory cytokines in rat primary macrophages in vitro and in vivo in BALB/c mice with greater efficacy than TempL. The results showed that a simple amino acid substitution in a short hydrophobic antimicrobial peptide, TempL, enhanced its antiendotoxin properties and illustrate a plausible correlation between its aggregation properties in LPS and LPS detoxification activity.

Topics: Amino Acid Substitution; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cells, Cultured; Cytokines; Endotoxins; Female; Humans; Hydrophobic and Hydrophilic Interactions; Inflammation; L Cells; Lipopolysaccharides; Lysine; Macrophages; Mice; Mice, Inbred BALB C; Proteins; Rats; Rats, Wistar; Structure-Activity Relationship