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Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects.

ABSTRACT: The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through its N-terminal W stretch, WWWKYE21 displays higher membrane binding, liposome rupture, and bacterial killing than unmodified KYE21. Analogously, W-tagging promotes binding to E. coli LPS and to its endotoxic lipid A moiety. Furthermore, WWWKYE21 causes more stable peptide/LPS complexes than KYE21, as evidenced by detailed NMR studies, adopting a pronounced helical conformation, with a large hydrophobic surface at the N-terminus due to the presence of W-residues, and a flexible C-terminus due to presence of several positively charged arginine residues. Mirroring its increased affinity for LPS and lipid A, WWWKYE21 displays strongly increased anti-inflammatory effect due to a combination of direct lipid A binding, peptide-induced charge reversal of cell membranes for LPS scavenging, and peptide-induced fragmentation of LPS aggregates for improved phagocytosis. Importantly, potent anti-inflammatory effects were observed at low cell toxicity, demonstrated for both monocytes and erythrocytes.

SUBMITTER: Singh S

PROVIDER: S-EPMC5427892 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects.

Singh Shalini S Datta Aritreyee A Schmidtchen Artur A Bhunia Anirban A Malmsten Martin M

Scientific reports 20170316 1

The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through i ...[more]

PMID: 28303012

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Project description:Context Torin2 has various pharmacological properties. However, its anti-inflammatory activity has not been reported. Objective This study focused on the potential anti-inflammatory properties of Torin2 in lipopolysaccharide (LPS)-evoked RAW264.7 murine macrophages. The study aimed to shed light on the molecular mechanisms that ameliorate these effects. Methods Torin2 was applied to 100 ng/mL lipopolysaccharide-induced RAW 264.7 macrophages in vitro. Nitric oxide (NO) levels were detected using the Griess reagent kit. Prostaglandin E2 (PGE2), pro-inflammatory cytokines interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Gene expression of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were tested using real-time quantitative polymerase chain reaction (qPCR). Cyclooxygenase-2 and inducible nitric oxide synthase proteins, phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, I-kappa-B-alpha (IκBα), and nuclear factor-kappa-B (NF-κB), and activation in extracts were detected via western blotting. Nuclear factor-kappa-B/p65 nuclear translocation was tested using an immunofluorescence assay. Results The results demonstrated that pre-treatment with Torin2 profoundly attenuated the lipopolysaccharide-stimulated levels of nitric oxide and prostaglandin E2, pro-inflammatory cytokines, messenger ribonucleic acid (mRNA), and protein expression of cyclooxygenase-2 and inducible nitric oxide synthase. Collectively, Torin2 pre-treatment notably weakened lipopolysaccharide-induced damage by reducing the phosphorylation of nuclear factor-kappa-B, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase proteins, and nuclear factor-kappa-B/p65 nuclear translocation. Conclusion Numerous pieces of evidence indicated that Torin2 reversed inflammatory activation by regulating nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways and provided a tentative potential candidate for preventing and treating inflammatory diseases. Torin2; RAW264.7 murine macrophages; Lipopolysaccharide; Anti-inflammation; Mechanisms.

Dataset Information

Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects.

Publications

Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects.

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