Phage-displayed antibodies for the detection of glycated proteasome in aging cells
Regina Gonzalez-Dosal1, Morten Draeby Sørensen, Brian F C Clark
1Danish Centre of Molecular Gerontology, Department of Molecular Biology, University of Aarhus, Denmark.
Abstract:
Accumulation of posttranslationally damaged proteins during aging could explain the decline of cell performance with age. N(epsilon)-carboxymethyllysine (CML) is the major glycation product on damaged proteins, causing dysfunction and cross-linking. The proteasome, a multicatalytic degradation complex, is one of the pathways for eliminating damaged proteins, and thus regulating their accumulation within the cell. However, the proteinase activities of the proteasome decline during aging. This may be due to posttranslational modifications of the subunits forming the proteasome complex. Using phage display technology, we have selected 16 single-chain variable fragments (scFv) recognizing the CML-modified alpha7 subunit of the proteasome. Using one of them, Ab3, we have observed a five-fold increase of CML-alpha7 in old human skin fibroblasts in comparison with young fibroblasts and telomerase-immortalized bone marrow cells (hTERT-BMCs).
Insights
Protein damage, marked by N(epsilon)-carboxymethyllysine (CML), increases with age. Researchers found elevated CML-modified proteasome subunits in aged cells, suggesting a link between protein damage and cellular aging.
Area of Science:
- Biochemistry
- Cell Biology
- Aging Research
Background:
- Accumulation of damaged proteins, like N(epsilon)-carboxymethyllysine (CML)-modified proteins, contributes to cellular dysfunction during aging.
- The proteasome degrades damaged proteins, but its activity declines with age, potentially due to subunit modifications.
- Posttranslational modifications of proteasome subunits may impair its function in aged cells.
Purpose of the Study:
- To investigate the presence and accumulation of damaged proteins within the proteasome during aging.
- To identify specific proteasome subunits modified by N(epsilon)-carboxymethyllysine (CML).
- To develop tools for quantifying CML-modified proteasome subunits in aged cells.
Main Methods:
- Phage display technology was employed to select single-chain variable fragments (scFv) targeting CML-modified proteins.
- Specific scFv, named Ab3, was generated to recognize the CML-modified alpha7 subunit of the proteasome.
- Quantitative analysis of CML-alpha7 levels was performed in aged and young human skin fibroblasts and hTERT-BMCs.
Main Results:
- Sixteen scFv clones recognizing CML-modified proteasome subunits were successfully selected.
- The scFv Ab3 demonstrated high specificity for the CML-modified alpha7 proteasome subunit.
- A significant five-fold increase in CML-alpha7 was observed in old human skin fibroblasts compared to young cells and hTERT-BMCs.
Conclusions:
- N(epsilon)-carboxymethyllysine (CML) modification of the proteasome alpha7 subunit increases with cellular aging.
- The developed scFv Ab3 serves as a valuable tool for detecting CML-modified proteasome subunits.
- These findings suggest a mechanism linking protein damage and proteasome dysfunction to cellular aging.
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