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Updated: Oct 16, 2026
Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans
Published on: September 7, 2021
Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without
E Tarcsa1, G Szymanska, S Lecker
1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.
Insights
Damaged proteins, like aged calmodulin, are degraded by the proteasome. This process can occur directly without ubiquitination, especially when calmodulin lacks calcium, revealing a new protein degradation pathway.
Area of Science:
- Cellular Biology
- Biochemistry
- Protein Degradation
Background:
- The ubiquitin-proteasome pathway is crucial for degrading damaged proteins in eukaryotic cells.
- Calmodulin (CaM), vital for cell regulation, can undergo age-induced damage, acquiring isoaspartyl residues.
- This damage affects CaM's Ca(2+)-binding regions, potentially impacting its stability and degradation.
Purpose of the Study:
- To investigate the degradation mechanism of damaged calmodulin (CaM) via the ubiquitin-proteasome pathway.
- To determine if ubiquitination is a prerequisite for the proteasomal degradation of aged CaM.
- To explore the role of calcium ions (Ca(2+)) in the degradation and ubiquitination of native and aged CaM.
Main Methods:
- Microinjection of native and in vitro aged CaM into Xenopus oocytes.
- Analysis of CaM degradation rates in HeLa cell extracts.
- Assessment of CaM ubiquitination and degradation by purified muscle 26 S proteasomes.
- Investigation of Ca(2+) effects on CaM ubiquitination and proteolysis.
Main Results:
- In vitro aged CaM was degraded significantly faster than native CaM in Xenopus oocytes and HeLa cell extracts.
- Proteasomal degradation of aged CaM occurred independently of rapid ubiquitination.
- Calcium ions (Ca(2+)) promoted CaM ubiquitination but inhibited degradation, suggesting a dissociation between these processes.
- Purified 26 S proteasomes degraded both aged and native Ca(2+)-free CaM without ubiquitination.
- Ca(2+) binding protected native CaM from degradation more effectively than aged CaM, which often retained a degradable conformation.
Conclusions:
- Protein degradation by the proteasome can occur directly, without prior ubiquitination, particularly for conformationally flexible or damaged proteins.
- Calmodulin's degradation is regulated by Ca(2+) binding, which induces a non-degradable conformation in native CaM.
- Age-induced damage or ligand absence can lead to flexible CaM conformations that are directly targeted for proteasomal degradation.
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