title: Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.
abstract: The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and β-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and β-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.
authors.affiliation: Department of Pharmacy, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Di
authors.initials: D
authors.lastname: Li
authors.forename: Yao
authors.initials: Y
authors.lastname: Liu
authors.forename: Ya
authors.initials: Y
authors.lastname: Yang
authors.forename: Jian-hong
authors.initials: JH
authors.lastname: Chen
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Lin-yun
authors.initials: LY
authors.lastname: Zou
authors.forename: Zhi-qiang
authors.initials: ZQ
authors.lastname: Tian
authors.forename: Jun
authors.initials: J
authors.lastname: Lv
authors.forename: Pei-yuan
authors.initials: PY
authors.lastname: Xia
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D060829:Arthropod Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000276:immunology / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism*; D014404:Tubulin / Q000276:immunology / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D008565:Membrane Proteins; D010456:Peptides, Cyclic; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014404:Tubulin; C034705:antilipopolysaccharide factor (Limulus)