Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways

Methods in Molecular Biology
Zhe ShaA L Goldberg

Abstract

In certain physiological or pathological states (e.g., starvation, heat shock, or muscle atrophy) and upon drug treatments, the overall rate of protein degradation in cells may increase or decrease. These adaptations and pathological responses can occur through alterations in substrate flux through the ubiquitin-proteasome pathway (UPP), the autophagy-lysosomal system, or both. Therefore, it is important to precisely measure the activities of these degradation pathways in degrading cell proteins under different physiological states or upon treatment with drugs. In particular, proteasome inhibitors have become very important agents for treating multiple myeloma and very useful tools in basic research. To evaluate rigorously their efficacy and the cellular responses to other inhibitors, it is essential to know the degree of inhibition of protein breakdown. Unfortunately, commonly used assays of the activities of the UPP or autophagy rely on qualitative, indirect approaches that do not directly reflect the actual rates of protein degradation by these pathways. In this chapter, we describe isotopic pulse-chase methods to directly measure overall rates of protein degradation in cells by radiolabeling cell proteins and following thei...Continue Reading

Citations

Mar 15, 2019·Pharmacological Reviews·Tiffany A Thibaudeau, David M Smith
Mar 4, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Mayara C F GewehrEmer S Ferro
Feb 21, 2019·Proceedings of the National Academy of Sciences of the United States of America·Jordan J S VerPlankAlfred L Goldberg
Jun 28, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Rachel A Coleman, Darci J Trader
Aug 21, 2020·Proceedings of the National Academy of Sciences of the United States of America·Zhe Sha, Alfred L Goldberg
Jun 3, 2021·Biomolecules·Alfred L GoldbergGalen Andrew Collins

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