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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetylcysteine / analogs & derivatives*
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Acetylcysteine / pharmacology
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Acute-Phase Proteins*
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Animals
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Carrier Proteins / metabolism*
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Cell Line
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Chymotrypsin / metabolism
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Cross-Linking Reagents / chemical synthesis
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Cysteine Endopeptidases / metabolism*
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Cysteine Proteinase Inhibitors / pharmacology
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Down-Regulation / drug effects
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Down-Regulation / genetics
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Down-Regulation / immunology
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Enzyme Activation / immunology
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Escherichia coli / chemistry
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Escherichia coli / genetics
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Glutamate Synthase / metabolism
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Leupeptins / pharmacology
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Lipopolysaccharides / administration & dosage
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Lipopolysaccharides / antagonists & inhibitors
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Lipopolysaccharides / chemical synthesis
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Lipopolysaccharides / metabolism*
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Lipopolysaccharides / pharmacology*
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Macrophages / drug effects
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Macrophages / enzymology*
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Macrophages / immunology
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Macrophages / metabolism
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Macrophages, Peritoneal / drug effects
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Macrophages, Peritoneal / enzymology
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Macrophages, Peritoneal / immunology
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Macrophages, Peritoneal / metabolism
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Membrane Glycoproteins*
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Membrane Proteins / metabolism
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Methanosarcina / enzymology
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Mice
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Mice, Inbred C3H
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Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinases / metabolism
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Multienzyme Complexes / antagonists & inhibitors*
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Multienzyme Complexes / metabolism*
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Phosphorylation / drug effects
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Proteasome Endopeptidase Complex
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Shock, Septic / immunology
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Shock, Septic / mortality
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Shock, Septic / prevention & control
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Signal Transduction / drug effects
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Signal Transduction / immunology*
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Tritium / metabolism
Substances
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Acute-Phase Proteins
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Carrier Proteins
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Cross-Linking Reagents
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Cysteine Proteinase Inhibitors
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Leupeptins
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Lipopolysaccharides
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Membrane Glycoproteins
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Membrane Proteins
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Multienzyme Complexes
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Re lipopolysaccharide
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lipopolysaccharide-binding protein
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Tritium
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lactacystin
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Glutamate Synthase
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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Chymotrypsin
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde
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Acetylcysteine