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Measurement of the Multiple Activities of 26S Proteasomes - PubMed

Measurement of the Multiple Activities of 26S Proteasomes

Hyoung Tae Kim et al. Methods Mol Biol. 2018.

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.

Keywords: 26S proteasomes; ATP hydrolysis; Deubiquitination; Gate opening; Peptide hydrolysis; Ubiquitin binding; Ubiquitin conjugate degradation; Ubiquitin-proteasome pathway.

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Figures

Figure 1.
Figure 1.

Stimulation of peptidase activities of purified mouse embryonic fibroblast 26S proteasomes by ATPγS. Activities were assayed by measuring hydrolysis of fluorogenic peptide-amc substrates (10 μM) in the presence of 100 μM ATP or ATPγS. The markedly stimulated hydrolysis of all three substrate in the presence of ATPλS over the basal activities indicates gate-opening induced by the non-hydrolysable nucleotide. The basal peptidase activity of the proteasomes was measured in the presence of ATP. Peptidase activities are presented as the Means ± SD of at least 3 independent measurements. (Modified from Kim and Goldberg, 2017(38))

Figure 2.
Figure 2.

Disassembly of K63 tetra Ub-chain (368 nM) by WT 26S proteasomes ones and lacking Usp14 (Usp14KO) was assayed after incubation at 37ºC for 20 min. Proteasomes were purified from mouse embryonic fibroblast. The generation of tri-, di- and mono-Ubs was analyzed by Western blotting after electrophoresis. Usp14KO 26S proteasomes disassembled tetra-Ub chains much faster than WT 26S particles. Although Ub-vinylsulfone (Ub-VS, 1.5 μM) did not inhibit Ub chain hydrolysis, o-phenanthroline (OPT, 1 mM) inhibits markedly and reduces deubiquitination by Usp14KO 26S to the level of WT 26S. Thus, the faster disassembly of Ub chains by the Usp14KO 26S involves the Zn2+-metalloprotease Rpn11 and not Uch37. (Adopted data from Kim and Goldberg, 2017(38))

Figure 3.
Figure 3.

ATP hydrolysis by mouse embryonic fibroblast 26S proteasomes was measured using a modified Malachite-Green assay (46). Upon addition of casein (1 μM) and a hexa-Ub chain (1 μM), which together mimic the binding of a Ub conjugate (37), ATP hydrolysis of WT 26S increased 2–3 fold. However, hexa-Ub alone or casein alone did not increase ATP hydrolysis. 6Ub: a linear hexa-Ub chain. Data are represented as means ± SD. (Modified from Kim and Goldberg, 2017(38))

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