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Measurement of the Multiple Activities of 26S Proteasomes

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Book cover The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

Abstract

Because proteasomes catalyze most of the protein degradation in mammalian cells, and their functioning is essential for cellular homeostasis, proteasome structure, biochemical mechanisms, and regulation in normal and disease states are now widely studied and are of major importance. In addition, inhibitors of the proteasome’s peptidase activity have proven to be very valuable as research tools and in the treatment of hematologic malignancies, and a number of newer pharmacological agents that alter proteasome function are being developed. The rapid degradation of ubiquitinated proteins by the 26S proteasome involves multiple enzymatic and non-enzymatic steps, including the binding of ubiquitinated substrates to the 19S particle (Subheading 3.2), opening the gated substrate entry channel into the 20S particle (Subheading 3.3), disassembly of the Ub chain (Subheading 3.4), ATP hydrolysis (Subheading 3.5), substrate unfolding and translocation, and proteolysis within the 20S particle (Subheadings 3.3 and 3.7). Assaying each of these processes is important if we are to fully understand the physiological regulation of proteasome function and the effects of disease or drugs. Here, we describe several methods that we have found useful to measure many of these individual activities using purified proteasomes. Studies using these approaches have already provided valuable new insights into the effects of post-synthetic modifications to 26S subunits, the physiological regulation of the ubiquitin-proteasome system, and the impairment of proteasome activity in neurodegenerative disease. These advances would not have been possible if only the standard assays of peptidase activity were used.

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Acknowledgments

The methods described here were developed through research support to our laboratory from the NIH-NIGMS (R01 GM51923), Cure Alzheimer's Fund, Muscular Dystrophy Association (MDA-419143), and Project A.L.S (2015-06).

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Author notes

  1. Hyoung Tae Kim and Galen Andrew Collins contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Hyoung Tae Kim, Galen Andrew Collins & Alfred L. Goldberg

Authors
  1. Hyoung Tae Kim
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  2. Galen Andrew Collins
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  3. Alfred L. Goldberg
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Corresponding author

Correspondence to Alfred L. Goldberg .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

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Kim, H.T., Collins, G.A., Goldberg, A.L. (2018). Measurement of the Multiple Activities of 26S Proteasomes. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_19

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8705-4

  • Online ISBN: 978-1-4939-8706-1

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