Site-specific methionine oxidation initiates calmodulin degradation by the 20S proteasome.
The proteasome is a key intracellular protease that regulates processes, such as signal transduction and protein quality control, through the selective degradation of specific proteins. Signals that target a protein for degradation, collectively known as degrons, have been defined for many proteins involved in cell signaling. However, the molecular signals involved in recognition and degradation of proteins damaged by oxidation have not been completely defined. The current study used biochemical and spectroscopic measurements to define the properties in calmodulin that initiate degradation by the 20S proteasome. Our experimental approach involved the generation of multiple calmodulin mutants with specific Met replaced by Leu. This strategy of site-directed mutagenesis permitted site-selective oxidation of Met to Met sulfoxide. We found that the oxidation-induced loss of secondary structure, as measured by circular dichroism, correlated with the rate of degradation for wild-type and mutants containing Leu substitutions in the C-terminus. However, no degradation was observed for mutants with Met to Leu substitution in the N-terminus, suggesting that oxidation-induced structural unfolding in the N-terminal region is essential for degradation by the 20S proteasome. Experiments comparing the thermodynamic stability of CaM mutants helped to further localize the critical site of oxidation-induced focal disruption between residues 51 and 72 in the N-terminal region. This work brings new biochemical and structural clarity to the concept of the degron, the portion of a protein that determines its susceptibility to degradation by the proteasome.
Balog
Edward M
EM
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Lockamy
Elizabeth L
EL
Thomas
David D
DD
Ferrington
Deborah A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D034421:Sus scrofa; D013816:Thermodynamics
D002147:Calmodulin; D050505:Mutant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D008715:Methionine; D046988:Proteasome Endopeptidase Complex