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Activation of the cell death program by nitric oxide involves inhibition of the proteasome
S Glockzin1, A von Knethen, M Scheffner
1Faculty of Medicine, Department of Medicine IV-Experimental Division, University of Erlangen-Nürnberg, Loschgestrasse 8, 91054 Erlangen, Germany.
Abstract:
The ubiquitin/proteasome pathway mediates the degradation of many short-lived proteins that are critically involved in the regulation of cell proliferation and cell death, including the tumor suppressor protein p53. Accumulation of p53 and induction of apoptosis in RAW 264.7 macrophages in response to nitric oxide are well established. However, the molecular mechanisms involved in nitric oxide-induced p53 accumulation are unknown. Here we show that, similar to nitric oxide, treatment of macrophages with specific proteasome inhibitors, including clastolactacystin-beta-lactone, induces p53 accumulation and apoptosis, suggesting that nitric oxide may affect the activity of the proteasome. In support of this hypothesis, both exposure of cells to S-nitrosoglutathione and stimulation of endogenous nitric oxide production by lipopolysaccharide/interferon-gamma treatment result in inhibition of proteasome activity as measured in vitro by the degradation of the proteasome-specific substrate succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide. Moreover, chemically diverse nitric oxide donors interfere with proteasome-mediated degradation of polyubiquitinated p53 in vitro. These data imply that nitric oxide-induced apoptosis and accumulation of p53 are, at least in part, mediated by inhibition of the proteasome.
Insights
Nitric oxide accumulation of the tumor suppressor p53 and apoptosis in macrophages is mediated by proteasome inhibition. This study reveals nitric oxide
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- The ubiquitin/proteasome pathway degrades short-lived proteins, including tumor suppressor p53.
- Nitric oxide (NO) is known to induce p53 accumulation and apoptosis in macrophages.
- The precise molecular mechanisms behind NO-induced p53 accumulation remain unclear.
Purpose of the Study:
- To investigate the molecular mechanisms underlying nitric oxide-induced p53 accumulation and apoptosis in macrophages.
- To determine if nitric oxide affects proteasome activity.
Main Methods:
- Treatment of macrophages with proteasome inhibitors and nitric oxide donors.
- Measurement of proteasome activity using a specific substrate (succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide).
- In vitro assessment of proteasome-mediated degradation of polyubiquitinated p53.
Main Results:
- Proteasome inhibitors and nitric oxide both induce p53 accumulation and apoptosis in macrophages.
- Nitric oxide exposure (S-nitrosoglutathione) and endogenous NO production inhibit proteasome activity.
- Nitric oxide donors interfere with the in vitro proteasome-mediated degradation of p53.
Conclusions:
- Nitric oxide-induced apoptosis and p53 accumulation in macrophages are, at least partially, mediated by proteasome inhibition.
- This study elucidates a novel mechanism linking nitric oxide signaling to proteasome function in the context of cell death and proliferation regulation.