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09:05
Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae
Published on: April 18, 2016
Two pathways for cyclooxygenase-2 protein degradation in vivo
Masayuki Wada1, Thomas L Saunders, Jason Morrow
1Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
The Journal of Biological Chemistry
|September 18, 2009
Summary
The endoplasmic reticulum-associated degradation pathway limits cyclooxygenase-2 (COX-2) levels in vivo. Deleting a key segment prevents this degradation, leading to elevated COX-2 and altered inflammatory responses.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Cyclooxygenase-2 (COX-2), or prostaglandin endoperoxide H synthase-2 (PGHS-2), is crucial for prostaglandin synthesis.
- COX-2 is implicated in inflammation and overexpressed in colon cancer.
- A specific 18-amino acid segment (residues 595-612) is vital for N-glycosylation and subsequent endoplasmic reticulum-associated degradation (ERAD) of COX-2.
Purpose of the Study:
- To investigate the in vivo significance of the ERAD pathway in regulating COX-2 protein levels.
- To characterize the physiological consequences of impaired COX-2 degradation.
Main Methods:
- Generation of a knock-in mouse model (Delta 18 COX-2) lacking the 18-amino acid segment required for ERAD.
- Analysis of renal, reproductive, and inflammatory phenotypes in Delta 18 COX-2 mice.
- Assessment of COX-2 expression in various tissues and cell types (brain, macrophages, fibroblasts) from knock-in and wild-type mice.
- Investigation of the effects of NSAIDs (flurbiprofen, ibuprofen) on COX-2 degradation.
Main Results:
- Delta 18 COX-2 mice lack the abnormalities seen in COX-2 null mice but exhibit elevated urinary prostaglandin E(2) metabolite levels.
- These mice show a more severe and prolonged febrile response to bacterial endotoxin.
- Overexpression of Delta 18 COX-2 protein was observed in brain, macrophages, and fibroblasts.
- NSAID treatment demonstrated that substrate turnover influences COX-2 degradation, and flurbiprofen attenuates it.
- Constant COX-2 levels in kidneys and spleens suggest compensatory mechanisms.
Conclusions:
- The endoplasmic reticulum-associated degradation pathway is a significant in vivo mechanism for controlling COX-2 levels.
- Impairment of this pathway leads to elevated COX-2 and altered inflammatory responses.
- Substrate turnover plays a role in COX-2 degradation, modulated by NSAIDs.
- Compensatory mechanisms exist to maintain COX-2 homeostasis in specific tissues like the kidney and spleen.
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