Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells

Abstract

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.

The HERC proteins: functional and evolutionary insights

“…On the other hand, the fine regulation of HERC5 during inflammation also points to this protein having an important say in this process. As has been seen, HERC5 disappears during the early phase of inflammation, which might lead to temporal substrate stabilization, only to reappear several hours later, presumably in order to contribute to the end phases of inflammation: this would be in accordance with the proved importance of ubiquitin-mediated protein degradation during the resolution of inflammation [76]. Finally, the regulation of HERC5 by p53 and Rb, together with its interaction with cyclin E and other cyclins [72], makes it appealing to think of a possible role for HERC5 in cell cycle progression.…”

Cytosolic Ubiquitin and Ubiquitylation Rates in Human Peripheral Blood Mononuclear Cells During Sepsis

“…To investigate whether the reduced conjugated ubiquitin during sepsis could be explained by a down-regulation of ubiquitin protein ligase systems, ubiquitylation rates were studied using a nonradioactive modification of the previously described method, using a biotinylated ubiquitin (ubiquitin b ) substrate instead of 125 I[CT]-ubiquitin (16). Figure 3A shows a representative blotting experiment with healthy donor lysate and Figure 3B shows the corresponding progress curve of ubiquitin conjugation to cytosolic proteins derived from densitometric analysis.…”

“…The total ubiquitylation rate of cytosolic proteins, as a result of the total ubiquitin-protein ligase activity (16), was measured as incorporation of biotinylated ubiquitin (ubiquitin b ) into the sum of cytosolic proteins. Incubation mixtures contained 25 mM Tris/HCl, 5 mM MgCl, 10 mM NaCl, 1 mM dithiothreitol, 1 mM ATP, 200 mg/mL ubiquitin b (Biomol, Plymouth Meeting, PA), and 1 mg/mL lysate at pH 8.…”

“…Heparinized blood was collected and immediately used for human peripheral blood mononuclear cells (PBMNC) isolation and cytosolic lysate preparation, as previously described (16). In brief, PBMNC were isolated by density centrifugation over a Ficoll (Biochrom, Berlin, Germany) density gradient (18).…”

“…Furthermore, there are no studies of ubiquitin system alterations in mononuclear cells. We previously described that the overall cytosolic ubiquitin protein ligation rate, as a result of the total ubiquitin protein ligase activity, is reduced in human peripheral blood mononuclear cells in an in vitro model of gram-negative sepsis, i.e., endotoxin (lipopolysaccharide, LPS) stimulation (16). To obtain initial insights into the regulation during sepsis, we investigated cytosolic levels of free and conjugated ubiquitin, along with the total cytosolic ubiquitylation rate in extracts.…”