Human LL-23

LL-23 is a natural peptide corresponding to the 23 N-terminal amino acid residues of human host defense cathelicidin LL-37. LL-23 demonstrated, compared to LL-37, a conserved ability to induce the chemokine MCP-1 in human peripheral blood mononuclear cells, a lack of ability to suppress induction of the pro-inflammatory cytokine TNF-α in response to bacterial lipopolysaccharides (LPS), and reduced antimicrobial activity. Heteronuclear multidimensional nuclear magnetic resonance (NMR) characterization of LL-23 revealed similar secondary structures and backbone dynamics in three membrane-mimetic micelles: SDS, dodecylphosphocholine (DPC), and dioctanoylphosphatidylglycerol. The NMR structure of LL-23 determined in perdeuterated DPC contained a unique serine that segregated the hydrophobic surface of the amphipathic helix into two domains. To improve our understanding, Ser9 of LL-23was changed to either Ala or Val on the basis of homologous primate cathelicidins. These changes made the hydrophobic surface of LL-23 continuous and enhanced antibacterial activity. While identical helical structures did not explain the altered activities, a reduced rate of hydrogen-deuterium exchange from LL-23 to LL-23A9 to LL-23V9 suggested a deeper penetration of LL-23V9 into the interior of the micelles, which correlated with enhanced activities. Moreover, these LL-23 variants had discrete immunomodulatory activities. Both restored the TNF-α dampening activity to the level of LL-37. Furthermore, LL-23A9, like LL-23, maintained superior protective MCP-1 production, while LL-23V9 was strongly immunosuppressive, preventing baseline MCP-1 induction and substantially reducing LPS-stimulated MCP-1 production. Thus, these LL-23 variants, designed on the basis of a structural hot spot, are promising immune modulators that are easier to synthesize and less toxic to mammalian cells than the parent peptide LL-37.

Purpose: It has been suggested that restoring gut microbiota alterations with probiotics represents a potential clinical target for the treatment of gut microbiota-related diseases, such as obesity. Here, we apply 16S rDNA microbiota profiling to establish which bacteria in the human gut are associated with obesity and cardiometabolic risk factors, and to evaluate whether probiotic supplementation modulates gut microbiota. Methods: We evaluated the effects of a probiotic mixture (2 × 1010 CFU/day of Lactobacillus acidophilus LA-14, Lactobacillus casei LC-11, Lactococcus lactis LL-23, Bifidobacterium bifidum BB-06, and Bifidobacterium lactis BL-4) in 32 overweight or obese women in a double-blind, randomized, placebo-controlled study. Using 16S rDNA sequencing, we characterized fecal samples and investigated the relationships between microbiome data and diet, body composition, antioxidant enzymes, and inflammatory profile. In addition, we characterized the degree of variation among fecal communities after the intervention. Results: BMI, weight, fat mass, lean mass, conicity index, protein intake, monounsaturated fat intake, glycated hemoglobin, TNF-α, and IL6/IL10 were significantly correlated with microbiome composition. The candidate division TM7 was strongly associated with all adiposity markers and Clostridiaceae associated negatively with TNF-α. The family Clostridiaceae increased and TM7 tended to decrease after the probiotic mixture supplementation. Subjects were clustered according to body composition, and a higher proportion of TM7 was observed in those with higher adiposity. Conclusions: Ecosystem-wide analysis of probiotic use effects on the gut microbiota revealed a genera specific influence, and one of which (TM7) represents a promising novel target for obesity treatment. Trial registration number: U1111-1137-4566.

Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 microM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.