Proteasome activity is required for anthrax lethal toxin to kill macrophages.
Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IkappaB-alpha degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.
Tang
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Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Leppla
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000633:toxicity*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D048369:MAP Kinase Kinase 1; D008264:Macrophages / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; C030325:anthrax toxin; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex