Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

Cundiff, Mary D.; Hurley, Christina M.; Wong, Jeremy D.; Bashyal, Aarti; Rosenberg, Jake; Reichard, Eden L.; Nassif, Nicholas D.; Brodbelt, Jennifer S.; Kraut, Daniel A.

doi:10.1101/592378

Cited by 3 publications

(6 citation statements)

References 65 publications

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“…Interestingly, mutations that interrupt this process can help in the discovery of an efficient way to inhibit the proteasome (electronic supplementary material, S1). Generally, the degradation process results in the formation of small peptides, which can be degraded to smaller amino acid chains by other processes [31].…”

Section: Protein Degradation Processmentioning

confidence: 99%

Ubiquitin–proteasome system and the role of its inhibitors in cancer therapy

et al. 2021

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Despite all the other cells that have the potential to prevent cancer development and metastasis through tumour suppressor proteins, cancer cells can upregulate the ubiquitin–proteasome system (UPS) by which they can degrade tumour suppressor proteins and avoid apoptosis. This system plays an extensive role in cell regulation organized in two steps. Each step has an important role in controlling cancer. This demonstrates the importance of understanding UPS inhibitors and improving these inhibitors to foster a new hope in cancer therapy. UPS inhibitors, as less invasive chemotherapy drugs, are increasingly used to alleviate symptoms of various cancers in malignant states. Despite their success in reducing the development of cancer with the lowest side effects, thus far, an appropriate inhibitor that can effectively inactivate this system with the least drug resistance has not yet been fully investigated. A fundamental understanding of the system is necessary to fully elucidate its role in causing/controlling cancer. In this review, we first comprehensively investigate this system, and then each step containing ubiquitination and protein degradation as well as their inhibitors are discussed. Ultimately, its advantages and disadvantages and some perspectives for improving the efficiency of these inhibitors are discussed.

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Section: Protein Degradation Processmentioning

confidence: 99%

Ubiquitin–proteasome system and the role of its inhibitors in cancer therapy

et al. 2021

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“…This extra unfolding ability is mediated, at least in part, by the Rpn13 ubiquitin receptor. We had previously proposed that binding of ubiquitinated proteins to Rpn13 might help activate the proteasome's unfolding ability by shifting the conformation from the s1 substrate-binding state to s3-like substrate processing states; in the kinetic model of Figure 4A this would correspond to increasing the initial engagement, degradation, and/or unfolding rates (16,25). The same hypothesis could explain the UBL versus Ub4 results we see, given that Ub4 substrates rely primarily on Rpn10, while UBL substrates rely primarily on Rpn13 (18).…”

Section: Discussionmentioning

confidence: 99%

“…Yeast (S. cerevisiae) proteasome was purified from wild type strain YYS40 or receptor mutant strains using a 3X-FLAG-tagged copy of Rpn11 subunit of the 19S particle as described previously (16,25). Cells were grown overnight in YEPD or selective media before being transferred to YEPD media for large scale growth at 30˚C.…”

Section: Proteasome Purificationmentioning

confidence: 99%

“…Neh2Dual-Barnase-DHFR substrate was expressed, purified, labeled and ubiquitinated as described previously (25). K63-linked chains were enzymatically synthesized and purified as described previously (16).…”

Section: Bacterial Protein Overexpression and Purificationmentioning

confidence: 99%

“…We recently showed that substrate ubiquitination can increase the proteasome's unfolding ability, and that proteasomal ubiquitin receptors mediate this increase, with Rpn13 playing the largest role (16,25). Herein we set out to determine if the degradation of GFP is be affected by the mode of targeting to the proteasome, and, if so, which ubiquitin receptors are responsible.…”

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Mode of Targeting to the Proteasome Determines GFP Fate

Bragança

Kraut

2020

Preprint

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The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasome’s ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

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Ubiquitin modulates 26 S proteasome conformational dynamics and promotes substrate degradation

Jönsson

Htet

Bard³

et al. 2022

Sci. Adv.

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The 26 S proteasome recognizes thousands of appropriate protein substrates in eukaryotic cells through attached ubiquitin chains and uses its adenosine triphosphatase (ATPase) motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Förster resonance energy transfer measurements to monitor the conformational dynamics of the proteasome, observe individual substrates during their progression toward degradation, and elucidate how these processes are regulated by ubiquitin chains. Rapid transitions between engagement- and processing-competent proteasome conformations control substrate access to the ATPase motor. Ubiquitin chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and aid substrate capture to accelerate degradation initiation. Upon substrate engagement, the proteasome remains in processing-competent states for translocation and unfolding, except for apparent motor slips when encountering stably folded domains. Our studies revealed how ubiquitin chains allosterically regulate degradation initiation, which ensures substrate selectivity in a crowded cellular environment.

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Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

Cited by 3 publications

References 65 publications

Ubiquitin–proteasome system and the role of its inhibitors in cancer therapy

Ubiquitin–proteasome system and the role of its inhibitors in cancer therapy

Mode of Targeting to the Proteasome Determines GFP Fate

Ubiquitin modulates 26 S proteasome conformational dynamics and promotes substrate degradation

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