Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells.
The ubiquitin-proteasome pathway is regarded as playing a crucial role in protein breakdown in inflammation and sepsis as well as in the regulation of inflammatory cell responses. In this pathway, ubiquitylation of target proteins is believed to act as a recognition signal for degradation by the 26S proteasome. As yet neither the ubiquitylation rate of cytosolic proteins, as a result of the total ubiquitin-protein ligase (tUbPL) activity, nor the specific ubiquitylation of calmodulin (ubiquitin-calmodulin ligase, uCaM-synthetase) has been determined in human mononuclear cells. Therefore, we studied cytosolic protein ubiquitylation in normal and in endotoxin (LPS)-stimulated human peripheral blood mononuclear cells (PBMNCs).PBMNCs from healthy volunteers were incubated with 0 or 100 ng/ml LPS for 18 h. Cytosolic extracts were obtained by hypotonic lysis and ultracentrifugation. TUbPL was measured as [(125)I]-[CT]-ubiquitin incorporation into the sum of cytosolic proteins. UCaM-synthetase activity was quantified with the fluphenazine (FP)-Sepharose affinity adsorption test. Endotoxin stimulation appears to inhibit tUbPL 3.7 +/- 2.7-fold to 48 +/- 43 fkat/mg (n = 6). UCaM-synthetase in cultures (n = 5) without endotoxin was determined to be 91 +/- 32 fkat/mg +Ca(2+) and 29 +/- 23 fkat/mg -Ca(2+). With endotoxin uCaM-synthetase was 138 +/- 73 fkat/mg +Ca(2+) and 14 +/- 22 fkat/mg -Ca(2+). Ca(2+)-specificity (ratio +/- Ca(2+)) of uCaM-synthetase increases from 3.1 without LPS to 10 after LPS stimulation, which was caused by a 2-fold decrease in minus Ca(2+) activity and a 1.5-fold increase in plus Ca(2+) activity. The data indicate specific regulatory effects of endotoxin on the cytosolic ubiquitylation systems in human PBMNCs.
Majetschak
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Klinik für Unfallchirurgie, Universitätsklinikum Mannheim, Fakultät für Klinische Medizin der Universität Heidelberg, Germany. matthias.majetschak@uch.ma.uni-heidelberg.de
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Häsler
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Obertacke
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D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D003546:Cysteine Endopeptidases / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity*; D009097:Multienzyme Complexes / Q000097:blood; D010453:Peptide Synthases / Q000097:blood; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000097:blood*
D001798:Blood Proteins; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases; D002118:Calcium