A Sensitive High‐Throughput Screening Method for Identifying Small Molecule Stimulators of the Core Particle of the Proteasome

Coleman, Robert W.; Trader, Darci J.

doi:10.1002/cpch.52

Cited by 5 publications

(8 citation statements)

References 17 publications

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“…HTS have been the preferred method of hit-to-lead campaigns. Trader developed a fluorescence resonance energy transfer (FRET) reporter assay for the HTS of proteasome activators [243,244]. The much larger size of the FRET reporter lowered the basal rate of hydrolysis and provided higher sensitivity and increased dynamic range over the field standard peptides.…”

Section: Proteasome Activatorsmentioning

confidence: 99%

Proteasome Activation to Combat Proteotoxicity

Jones

Tepe

2019

Molecules

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Loss of proteome fidelity leads to the accumulation of non-native protein aggregates and oxidatively damaged species: hallmarks of an aged cell. These misfolded and aggregated species are often found, and suggested to be the culpable party, in numerous neurodegenerative diseases including Huntington’s, Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), and Alzheimer’s Diseases (AD). Many strategies for therapeutic intervention in proteotoxic pathologies have been put forth; one of the most promising is bolstering the efficacy of the proteasome to restore normal proteostasis. This strategy is ideal as monomeric precursors and oxidatively damaged proteins, so called “intrinsically disordered proteins” (IDPs), are targeted by the proteasome. This review will provide an overview of disorders in proteins, both intrinsic and acquired, with a focus on susceptibility to proteasomal degradation. We will then examine the proteasome with emphasis on newly published structural data and summarize current known small molecule proteasome activators.

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Section: Proteasome Activatorsmentioning

confidence: 99%

Proteasome Activation to Combat Proteotoxicity

Jones

Tepe

2019

Molecules

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“…Our laboratory has previously shown success in developing probes capable of monitoring proteasome activity in cells. 16,[28][29][30] These probes have been shown to be sensitive enough to monitor both 20S CP inhibition and stimulation in cell models. Here, we utilize TAS3 (Figure 3A), a selective proteasome activity reporter, to determine if our small molecule stimulators are capable of increasing 20S CP activity in cells.…”

Section: A B Cmentioning

confidence: 99%

“…The most highly utilized cellular pathway to degrade unwanted or damaged proteins is the ubiquitin-dependent proteasome system (UPS). 1,2 For this degradation pathway, proteins are tagged with a chain of ubiquitin, a degradation signaling protein, which is recognized by the 26S proteasome (Figure 1A). The regulatory particle of the proteasome, commonly referred to as the 19S RP, is responsible for recognizing the ubiquitinated proteins, removing the ubiquitin moieties from the protein, and denaturing the structure of the protein so it can more easily fit into the core particle, or the 20S CP, to be hydrolyzed.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we sought to answer this question using several small molecule stimulators that have been shown to affect 20S CP activity to various degrees in our FRET assay. 21 Using these stimulators, we analyzed their effect on the 20S CP-mediated degradation of 15 different purified proteins to determine what types of proteins are more rapidly turned over in the presence of various 20S CP stimulators.…”

Section: Introductionmentioning

confidence: 99%

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Protein Degradation Profile Reveals Dynamic Nature of 20S CP Small Molecule Stimulation

Coleman¹,

Tj²,

Aryal³

et al. 2020

Preprint

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<p>Small molecules have recently been discovered to stimulate the 20S core particle (CP) of the proteasome to degrade proteins, such as a-synuclein. While these studies have focused on particular proteins that are known 20S CP substrates, it is currently unclear how many or what types of proteins may be affected by enhancing this degradation process. We present here a study that utilizes four 20S CP stimulators to determine how each can affect the degradation of proteins in a biochemical assay with purified proteins, an overexpressed GFP-fusion protein in cells, and the effects of stimulators using label-free quantitative proteomic analysis for a more broad understanding on their impact. The results of these studies highlight that the impact of small molecule stimulators of the 20S CP on protein degradation cannot be easily predicted. While 20S CP stimulators will likely increase the degradation of proteins that have significant disorder, such as a-synuclein and tau, we observed different impacts on the degradation of proteins less than 90% disordered. To gain greater insight into the cellular systems that may be affected by 20S CP stimulators, we analyzed the proteome of HEK-293T cells treated with two of our stimulators, AM-404 or miconazole. These results were then compared to cells treated with a proteasome inhibitor, bortezomib. Our results show that 20S CP stimulators affect a smaller number of proteins as compared to bortezomib. The proteomics analysis corroborates our biochemical and GFP-fusion protein results, confirming that the impact of a 20S CP stimulator on protein degradation is dependent on the stimulator. Taken together, this study reveals the dynamic nature of the 20S CP, as small molecule stimulators can have a variety of mechanisms of action to change protein degradation activity. Our studies show that 20S CP stimulation can lead to a decrease in protein levels, more so those proteins that are significantly disordered, and that small molecule stimulators can potentially be tailored to decrease certain protein types.</p>

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“…To answer this question, we chose to evaluate several small molecule stimulators that have been shown to affect 20S CP activity to various degrees in our FRET assay. 25 Using these stimulators, we analyzed their effect on the 20S CP-mediated degradation of 15 different purified proteins to determine what types of proteins are more rapidly turned over in the presence of various 20S CP stimulators. We then utilized a fluorescent peptide probe and GFP-fusion proteins to demonstrate the efficacy of our small molecule stimulators in HEK-293T cells.…”

Section: Introductionmentioning

confidence: 99%