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Cytotoxic frog antimicrobial peptide Temporin L (TempL) is an attractive molecule for the design of lead antimicrobial agents due to its short size and versatile biological activities. However, noncytotoxic TempL variants with desirable biological activities have rarely been reported. TempL analogue Q3K,TempL is water-soluble and possesses a significant antiendotoxin property along with comparable cytotoxicity to TempL. A phenylalanine residue, located at the hydrophobic face of Q3K,TempL and the “d” position of its phenylalanine zipper sequence, was replaced with a cationic lysine residue. This analogue, Q3K,F8K,TempL, showed reduced hydrophobic moment and was noncytotoxic with lower antimicrobial activity. Interestingly, swapping between tryptophan at the fourth and serine at the sixth positions turned Q3K,F8K,TempL totally amphipathic as reflected by its helical wheel projection with clusters of hydrophobic and hydrophilic residues and the highest hydrophobic moment among these peptides. Surprisingly, this analogue, SW,Q3K,F8K,TempL, was as noncytotoxic as Q3K,F8K,TempL but showed augmented antimicrobial and antiendotoxin properties, comparable to that of TempL and Q3K,TempL. SW,Q3K,F8K,TempL exhibited appreciable survival of mice against P. aeruginosa infection and a lipopolysaccharide (LPS) challenge. Unlike TempL and Q3K,TempL, SW,Q3K,F8K,TempL adopted an unordered secondary structure in bacterial membrane mimetic lipid vesicles and did not permeabilize them or depolarize the bacterial membrane. Overall, the results demonstrate the design of a nontoxic TempL analogue that possesses clusters of hydrophobic and hydrophilic residues with impaired secondary structure and shows a nonmembrane-lytic mechanism and in vivo antiendotoxin and antimicrobial activities. This paradigm of design of antimicrobial peptide with clusters of hydrophobic and hydrophilic residues and high hydrophobic moment but low secondary structure could be attempted further.

Vaccination is devised/formulated to stimulate specific and prolonged immune responses for long-term protection against infection or disease. A vaccine component, namely adjuvant, enhances antigen recognition by the host immune system and thereby stimulates its cellular and adaptive responses. Especially synthetic Toll-like receptor (TLR) agonists having selfassembling properties are considered as good candidates for adjuvant development. Here, a human TLR4-derived 20-residue peptide (TR-433), present in the dimerization interface of the TLR4 -myeloid differentiation protein-2 (MD2) complex, displayed self-assembly and adopted a nanostructure. Both in vitro studies and in vivo experiments in mice indicated that TR-433 is nontoxic. TR-433 induced pro-inflammatory responses in THP-1 monocytes and HEK293T cells that were transiently transfected with TLR4/CD14/MD2 and also in BALB/c mice. In light of the self-assembly and pro-inflammatory properties of TR-433, we immunized with a mixture of TR-433 and either ovalbumin or filarial antigen trehalose-6-phosphate phosphatase (TPP). A significant amount of IgG titers was produced, suggesting adjuvanting capability of TR-433 that was comparable with that of Freund's complete adjuvant (FCA) and appreciably higher than that of alum. We found that TR-433 preferentially activates type 1 helper T cell (T h 1) response rather than type 2 helper T cell (T h 2) response. To our knowledge, this is the first report on the identification of a short TLR4-derived peptide that possesses both self-assembling and pro-inflammatory properties and has significant efficacy as an adjuvant, capable of activating cellular responses in mice. These results indicate that TR-433 possesses significant potential for development as a new adjuvant in therapeutic application. 2 The abbreviations used are: FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; TLR, Toll-like receptor; LPS, lipopolysaccharide; TPP, trehalose-6-phosphate phosphatase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TEM, transmission EM; ThT, thioflavin T; TNF, tumor necrosis factor; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; Ova, ovalbumin; HRP, horseradish peroxidase; IFN, interferon. Figure 8. TR-433 adjuvant activity depends on T-bet expression. mRNA expressions of T-bet and GATA-3 were analyzed in splenocytes in duplicates in two independent experiments (n ϭ 2). Mouse glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control, and relative -fold change was determined by the comparative ⌬CT method. Statistical analysis was carried out using one-way analysis of variance using Dunnett's test.

Adiponectin is a fat tissue-derived adipokine with beneficial effects against diabetes, cardiovascular diseases, and cancer. Accordingly, adiponectin-mimetic molecules possess significant pharmacological potential. Oligomeric states of adiponectin appear to determine its biological activity. We identified a highly conserved, 13-residue segment (ADP-1) from adiponectin's collagen domain, which comprises GG motifs and has one asparagine and two histidine residues that assist in oligomeric protein assembly. We therefore hypothesized that ADP-1 promotes oligomeric assembly and thereby mediates potential metabolic effects. We observed here that ADP-1 is stable in human serum and oligomerizes in aqueous environments. We also found that ADP-1 activates AMP-activated protein kinase (AMPK) in an adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1)-dependent pathway and stimulates glucose uptake in rat skeletal muscle cells (L6 myotubes). ADP-1-induced glucose transport coincided with ADP-1-induced biosynthesis of glucose transporter 4 and its translocation to the plasma membrane. ADP-1 induced an interaction between APPL1 and the small GTPase Rab5, resulting in AMPK phosphorylation, in turn leading to phosphorylation of p38 mitogen-activated protein kinase (MAPK), acetyl-CoA carboxylase, and peroxisome proliferator-activated receptor α. Similar to adiponectin, ADP-1 increased the expression of the adiponectin receptor 1 () gene. Of note, ADP-1 decreased blood glucose levels and enhanced insulin production in pancreatic β cells in db/db mice. Further, ADP-1 beneficially affected lipid metabolism by enhancing lipid globule formation in mouse 3T3-L1 adipocytes. To our knowledge, this is the first report on identification of a short peptide from adiponectin with positive effects on glucose or fatty acid metabolism.

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