Characterization of Tollip protein upon Lipopolysaccharide challenge.
Tollip protein serves as a suppressor of innate immunity signaling with unknown mechanism. In this report, we observed that Tollip preferentially bound with phosphatidylinositol-3-phosphate (PtdIns(3)P) and phosphatidylinositol-3,4,5-phosphate (PtdIns(3,4,5)P) in vitro. Mutation of lysine 150 to glutamic acid (Tollip(KE)) within the C2 domain abolished such binding, indicating that C2 domain is critically involved. We demonstrated that overexpression of Tollip inhibited NFkB reporter gene transcription. On the contrary, overexpression of Tollip(KE) mutant has no inhibitory effect on LPS-induced NFkB reporter activity. Tollip binding with 3'-phosphorylated phosphatidylinositides implies that PI3 kinase may regulate its function. We observed that Tollip-mediated inhibition could be alleviated by wortmannin. We also showed that pretreatment with wortmannin augmented LPS-induced endogenous IL-1beta gene expression in monocytic THP-1 cells. THP-1 cells with prolonged LPS treatment develop a state of hyporesponsiveness and no longer respond to further LPS challenge in terms of IL-1beta gene expression. Here we demonstrated that Tollip protein levels were increased following LPS treatment in THP-1 cells as well as in human primary blood mononuclear cells. Increased protein stability upon LPS challenge was likely the cause for the Tollip protein increase. Upon over expression with an enhanced green fluorescent tag, Tollip localized to Golgi apparatus. Our study provides yet another mechanism for suppressing excessive TLR signaling activation mediated by Tollip.
Li
Tao
T
Department of Medicine, Medical Center Blvd, 3045 Hanes Bldg., Wake Forest University School of Medicine, Winston Salem, NC 27157, USA.
Hu
Jean
J
Li
Liwu
L
D000730:Androstadienes / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D053592:Interleukin-1 Receptor-Associated Kinases; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; C410602:TOLLIP protein, human; C060974:phosphatidylinositol 3,4,5-triphosphate; C055525:phosphatidylinositol 3-phosphate; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D000077191:Wortmannin