Scalable In Vitro Proteasome Activity Assay

Gautam, Amit; Martinez‐Fonts, Kirby; Matouschek, Andreas T

doi:10.1007/978-1-4939-8706-1_21

Cited by 7 publications

(12 citation statements)

References 57 publications

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“…Fluorescence anisotropy is useful to follow 20S and 26S proteasome degradation of fluorescent dye–labeled protein substrates in real time (Bhattacharyya et al, 2016; Thibaudeau et al, 2018). Singh Gautam et al (2018) describe methods for high-throughput measurement of 26S ubiquitin-dependent degradation using dye-labeled substrates.…”

Section: Methods For Pharmacological Proteasome Researchmentioning

confidence: 99%

“…The 26S ubiquitin-dependent degradation of folded proteins can be monitored with a tetra-ubiquitin fused green fluorescent protein (GFP) (Martinez-Fonts and Matouschek, 2016; Singh Gautam et al, 2018) with a C-terminal unstructured region (Prakash et al, 2004) or a tetra-ubiquitinated dihydrofolate reductase (Thrower et al, 2000). It is also possible to express some cODC fusion proteins in vivo to monitor proteasome activity.…”

Section: Methods For Pharmacological Proteasome Researchmentioning

confidence: 99%

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A Practical Review of Proteasome Pharmacology

2019

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The ubiquitin proteasome system (UPS) degrades individual proteins in a highly regulated fashion and is responsible for the degradation of misfolded, damaged, or unneeded cellular proteins. During the past 20 years, investigators have established a critical role for the UPS in essentially every cellular process, including cell cycle progression, transcriptional regulation, genome integrity, apoptosis, immune responses, and neuronal plasticity. At the center of the UPS is the proteasome, a large and complex molecular machine containing a multicatalytic protease complex. When the efficiency of this proteostasis system is perturbed, misfolded and damaged protein aggregates can accumulate to toxic levels and cause neuronal dysfunction, which may underlie many neurodegenerative diseases. In addition, many cancers rely on robust proteasome activity for degrading tumor suppressors and cell cycle checkpoint inhibitors necessary for rapid cell division. Thus, proteasome inhibitors have proven clinically useful to treat some types of cancer, especially multiple myeloma. Numerous cellular processes rely on finely tuned proteasome function, making it a crucial target for future therapeutic intervention in many diseases, including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes, and cancer. In this review, we discuss the structure and function of the proteasome, the mechanisms of action of different proteasome inhibitors, various techniques to evaluate proteasome function in vitro and in vivo, proteasome inhibitors in preclinical and clinical development, and the feasibility for pharmacological activation of the proteasome to potentially treat neurodegenerative disease.

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Section: Methods For Pharmacological Proteasome Researchmentioning

confidence: 99%

Section: Methods For Pharmacological Proteasome Researchmentioning

confidence: 99%

A Practical Review of Proteasome Pharmacology

2019

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“…Ub4 binds ~4 times more weakly to the proteasome than UBL (15,19). However, decreasing the affinity 4-fold in our model has only a minor impact on simulated degradation, such that the proteasome should still largely degrade the substrate with minimal clipping.…”

Section: Rpn13 Is Primarily Responsible For Strong Unfolding With Ublmentioning

confidence: 88%

“…Extracting a single UBL domain may be easier than extracting Ub4, which might more easily re-bind during the process. However, the UBL domain binds ~4-fold more tightly to the proteasome than Ub4 (15,19), arguing against this model. It has long been puzzling why so many ubiquitin receptors and shuttle proteins were needed for proteasomal function.…”

Section: Discussionmentioning

confidence: 99%

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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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CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH

et al. 2023

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Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S) x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S) x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S) x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S) x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S) x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound endto-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

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Scalable In Vitro Proteasome Activity Assay

Cited by 7 publications

References 57 publications

A Practical Review of Proteasome Pharmacology

A Practical Review of Proteasome Pharmacology

Mode of Targeting to the Proteasome Determines GFP Fate

CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH

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