Reporter-Based Screens for the Ubiquitin/Proteasome System

Abstract

Instant and adequate handling of misfolded or otherwise aberrant proteins is of paramount importance for maintaining protein homeostasis in cells. The ubiquitin/proteasome system (UPS) is a central player in protein quality control as it operates in a seek-and-destroy mode, thereby facilitating elimination of faulty proteins. While proteasome inhibition is in clinical use for the treatment of hematopoietic malignancies, stimulation of the UPS has been proposed as a potential therapeutic strategy for various neurodegenerative disorders. High-throughput screens using genetic approaches or compound libraries are powerful tools to identify therapeutic intervention points and novel drugs. Unlike assays that measure specific activities of components of the UPS, reporter substrates provide us with a more holistic view of the general functional status of the UPS in cells. As such, reporter substrates can reveal new ways to obstruct or stimulate this critical proteolytic pathway. Here, we discuss various reporter substrates for the UPS and their application in the identification of key players and the pursuit for novel therapeutics.

Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

“…The effect of CBK77 was analyzed on additional reporters that represent different classes of proteasome substrates: ubiquitin-arginine-GFP (Ub-R-GFP), a soluble N-end rule substrate; YFP-CL1, an aggregation-prone substrate and lastly, a reporter based on the T cell receptor subunit CD3δ (CD3δ-YFP), which is degraded by ER-associated degradation (ERAD) [ 21 ]. Administration of CBK77, but not CBK07, resulted in accumulation of each of these substrates (Fig.…”

Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

“…ubiquitin-arginine-GFP (Ub-R-GFP), a soluble reporter targeted for proteasomal degradation by the N-end rule pathway; YFP-CL1, which carries a C-terminal degradation signal that renders the reporter aggregation-prone and lastly, a reporter based on the T cell receptor subunit CD3δ (CD3δ-YFP), which is degraded by ERassociated degradation (ERAD) 16 . Administration of CBK77, but not CBK07, to three MelJuSo cell lines stably expressing these UPS reporters resulted in accumulation of each of these substrates, suggesting that CBK77 has a general inhibitory effect on the UPS (Fig.…”

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

“…histolytica strain that express a GFP reporter substrate of UPS-mediated protein degradation. The ubiquitin-tagged GFP is cleaved in cells, and the free GFP undergoes ubiquitination by RING-E3 ubiquitin ligases for its subsequent degradation by the proteasome [ 50 ]. We found that mutant CSN5 overexpression disrupted protein degradation resulting in accumulation of GFP as measured by quantitative confocal imaging ( Fig 2F and 2G ) and fluorometric assay ( Fig 2H and S3H Fig ).…”