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 p rotein breakdown in inflammation and sepsis as well as in the regulation of inflammatory cell responses. In this pathway, ubiquitylation of target pro teins 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-s ynthetase) has been determined in human mononuclear cells. Therefore, we st udied cytosolic protein ubiquitylation in normal and in endotoxin (LPS)-sti mulated human peripheral blood mononuclear cells (PBMNCs). PBMNCs from heal thy volunteers were incubated with 0 or 100 ng/ml LPS for Is h, Cytosolic e xtracts were obtained by hypotonic lysis and ultracentrifugation, TUbPL was measured as [I-125]-[CT]-ubiquitin incorporation into the sum of cytosolic proteins. UCaM-synthetase activity was quantified with the fluphenazine (F P)-Sepharose affinity adsorption test. Endotoxin stimulation appears to inh ibit tUbPL 3.7 +/- 2.7-fold to 48 +/- 43 fkat/mg (n = 6). UCaM-synthetase i n cultures (n = 5) without endotoxin was determined to be 91 +/- 32 fkat/mg +Ca2+ and 29 +/- 23 fkat/mg -Ca2+. With endotoxin uCaM-synthetase was 138 +/- 73 fkat/mg +Ca2+ and 14 +/- 22 fkat/mg -Ca2+. Ca2+-specificity (ratio /- Ca2+) of uCaM-synthetase increases from 3.1 without LPS to 10 after LPS stimulation, which was caused by a 2-fold decrease in minus Ca2+ activity a nd a 1.5-fold increase in plus Ca2+ activity. The data indicate specific re gulatory effects of endotoxin on the cytosolic ubiquitylation systems in hu man PBMNCs.