M. Majetschak et al., Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells, J ENDOTOX R, 6(6), 2000, pp. 483-488
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.