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

24 April 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

Proteasome
Stimulator
20S CP

Supplementary materials

Title
Description
Actions
Title
Trader SI Final
Description
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.