title: The proteasome as a lipopolysaccharide-binding protein in macrophages: differential effects of proteasome inhibition on lipopolysaccharide-induced signaling events.
abstract: We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.
authors.affiliation: Department of Basic Medical Science, School of Medicine and Shock/Trauma Research Center, University of Missouri, Kansas City, MO 64108, USA. qureshi@umkc.edu
authors.forename: Nilofer
authors.initials: N
authors.lastname: Qureshi
authors.forename: Pin-Yu
authors.initials: PY
authors.lastname: Perera
authors.forename: Jing
authors.initials: J
authors.lastname: Shen
authors.forename: Guochi
authors.initials: G
authors.lastname: Zhang
authors.forename: Arnd
authors.initials: A
authors.lastname: Lenschat
authors.forename: Gary
authors.initials: G
authors.lastname: Splitter
authors.forename: David C
authors.initials: DC
authors.lastname: Morrison
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000209:Acute-Phase Proteins; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005970:Glutamate Synthase / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology; D051379:Mice; D008809:Mice, Inbred C3H; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex; D012772:Shock, Septic / Q000276:immunology / Q000401:mortality / Q000517:prevention & control; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D014316:Tritium / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000111:Acetylcysteine; D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D002918:Chymotrypsin; D003432:Cross-Linking Reagents; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D005970:Glutamate Synthase; D007976:Leupeptins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009097:Multienzyme Complexes; D046988:Proteasome Endopeptidase Complex; D014316:Tritium; D000099231:Lipopolysaccharide-Binding Protein; C085483:Re lipopolysaccharide; C067713:lactacystin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde