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Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation
J Gieche1, J Mehlhase, A Licht
1Neuroscience Research Center, Medical Faculty (Charité), Humboldt University Berlin, Schumannstr. 20/21, D-10098, Berlin, Germany.
Abstract:
Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.
Insights
Macrophages can increase protein turnover and oxidation following oxidative stress. They selectively remove oxidized proteins, protecting their intracellular protein pool from damage.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Macrophages produce reactive oxygen and nitrogen species during oxidative bursts.
- Oxidative stress leads to intracellular protein oxidation.
- The proteasomal system degrades oxidized proteins.
Purpose of the Study:
- To investigate if macrophages increase protein turnover and oxidation after oxidative stress.
- To determine if endogenous oxidant production affects protein turnover and oxidation.
- To elucidate the role of the proteasomal system in removing oxidized proteins in macrophages.
Main Methods:
- Utilized the mouse macrophage cell line RAW264.7.
- Exposed cells to hydrogen peroxide and peroxynitrite for oxidative stress.
- Activated cells with PMA to induce endogenous oxidant production.
- Employed the proteasome inhibitor lactacystin to study protein degradation pathways.
Main Results:
- Macrophage protein turnover and oxidation increased in response to oxidative stress.
- Endogenous oxidant production via PMA activation had similar effects.
- The proteasomal system was confirmed to selectively remove oxidized proteins.
Conclusions:
- Macrophages exhibit increased protein turnover and oxidation under oxidative stress.
- The proteasomal system plays a crucial role in the selective clearance of oxidized proteins.
- Macrophages possess mechanisms to protect their intracellular protein pool from oxidative damage.