ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
0/0

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

preprint
submitted on 23.04.2020 and posted on 24.04.2020 by Rachel A. Coleman, Tiago Jose Paschoal Sobreira, Uma Aryal, Darci Trader

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.

Funding

Cancer Center Support Grant (CORE) Renewal

National Cancer Institute

Find out more...

Development of Activity-Based Chemical Reporters to Differentiate Proteasome Isoforms in Cells

National Institute of General Medical Sciences

Find out more...

History

Email Address of Submitting Author

dtrader@purdue.edu

Institution

Purdue University

Country

United States

ORCID For Submitting Author

0000-0002-0607-1243

Declaration of Conflict of Interest

No conflict of interest

Exports

Logo branding

Exports