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Inhibition of the multicatalytic proteinase (proteasome) by 4-hydroxy-2-nonenal cross-linked protein
1Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Abstract:
Oxidative modification of glucose-6-phosphate dehydrogenase (Glu-6-PDH), as observed for other proteins, increases the susceptibility of the protein to degradation by the multicatalytic proteinase/proteasome (MCP). Oxidized Glu-6-PDH is, however, more prone to cross-linking reactions by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE), processes which render the protein resistant to proteolysis. In addition, HNE cross-linked protein inhibits the degradation of oxidatively modified glutamine synthetase by the MCP. In contrast to oxidized Glu-6-PDH, which inhibits the proteolysis of GS in a competitive manner, HNE cross-linked protein acts as a noncompetitive inhibitor. As judged by binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, a common structural feature of both macromolecular substrates and inhibitors of the MCP is an increased accessibility of hydrophobic regions on the protein.
Insights
Oxidative modification of glucose-6-phosphate dehydrogenase (Glu-6-PDH) can lead to its resistance to proteasome degradation. This modification, particularly by 4-hydroxy-2-nonenal (HNE), also inhibits other protein degradation pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Proteostasis
Background:
- Oxidative stress impacts protein stability and degradation.
- The multicatalytic proteinase/proteasome (MCP) is a key cellular machinery for protein turnover.
- Glucose-6-phosphate dehydrogenase (Glu-6-PDH) is susceptible to oxidative damage.
Purpose of the Study:
- To investigate the effect of oxidative modification on Glu-6-PDH.
- To determine the role of 4-hydroxy-2-nonenal (HNE) in modifying Glu-6-PDH.
- To elucidate the impact of modified Glu-6-PDH on proteasome function.
Main Methods:
- In vitro studies of protein oxidation and cross-linking.
- Analysis of protein susceptibility to degradation by the MCP.
- Enzyme kinetics to assess competitive and noncompetitive inhibition.
- Fluorescent probe binding to evaluate protein structural changes.
Main Results:
- Oxidative modification increases Glu-6-PDH susceptibility to MCP degradation.
- Lipid peroxidation product HNE cross-links oxidized Glu-6-PDH, conferring resistance to proteolysis.
- HNE-cross-linked Glu-6-PDH inhibits the degradation of oxidatively modified glutamine synthetase (GS) by the MCP.
- HNE-cross-linked protein acts as a noncompetitive inhibitor, unlike oxidized Glu-6-PDH (competitive inhibitor).
- Increased accessibility of hydrophobic regions is a common feature of MCP substrates and inhibitors.
Conclusions:
- HNE-mediated cross-linking of oxidized Glu-6-PDH alters its proteolytic fate and inhibitory properties.
- Modified proteins can interfere with proteasome function, impacting cellular proteostasis.
- Accessible hydrophobic regions are critical for protein recognition and interaction with the MCP.