Limited degradation of oxidized calmodulin by proteasome: formation of peptides
Miriam Strosova1, Peter Voss, Martina Engels
1Research Institute for Environmental Medicine gGmbH at the Heinrich-Heine-University, Duesseldorf, Germany.
Abstract:
Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H2O2 concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.
Insights
Oxidative stress causes protein damage, leading to calmodulin fragments. The 20S proteasome degrades oxidized calmodulin, producing specific peptides that resist further breakdown.
Area of Science:
- Biochemistry
- Cell Biology
- Proteostasis
Background:
- Oxidized proteins are preferentially degraded by the proteasome.
- Calmodulin (CaM) is a key calcium-binding protein affected by oxidative stress.
Purpose of the Study:
- To investigate the degradation of calmodulin under oxidative stress in human fibroblasts.
- To determine if CaM breakdown is oxidation-induced or proteasome-mediated.
Main Methods:
- Oxidative stress induction in human fibroblast cell lines.
- In vitro degradation assays using purified 20S proteasome and oxidized/unoxidized CaM.
- Analysis of CaM fragments and peptides via molecular weight and identification.
Main Results:
- Oxidative stress in fibroblasts generated low molecular weight CaM fragments.
- Native CaM was resistant to 20S proteasome degradation.
- Oxidized CaM was degraded by the 20S proteasome in a time- and H2O2-dependent manner.
- Identified peptides in stressed cells matched those from in vitro oxidized CaM degradation.
Conclusions:
- CaM fragmentation under oxidative stress is oxidation-driven.
- The 20S proteasome cleaves oxidized CaM into specific peptides.
- These peptides are resistant to further degradation, accumulating within cells.
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