Methods for the Discovery of Small Molecules to Monitor and Perturb the Activity of the Human Proteasome

Abstract

Regulating protein production and degradation is critical to maintaining cellular homeostasis. The proteasome is a key player in keeping proteins at the proper levels. However, proteasome activity can be altered in certain disease states, such as blood cancers and neurodegenerative diseases. Cancers often exhibit enhanced proteasomal activity, as protein synthesis is increased in these cells compared with normal cells. Conversely, neurodegenerative diseases are characterized by protein accumulation, leading to reduced proteasome activity. As a result, the proteasome has emerged as a target for therapeutic intervention. The potential of the proteasome as a therapeutic target has come from studies involving chemical stimulators and inhibitors, and the development of a suite of assays and probes that can be used to monitor proteasome activity with purified enzyme and in live cells.

Morpho functional features of proteasomes. Participation in pathological processes. Inhibitors of the ubiquitin-proteasome system as potential drugs

“…Each of the approaches has its own advantages and disadvantages. The main advantage of the affinity purification method is the speed of obtaining purified proteasomes: it takes several hours instead of several days [16].…”

“…Peptide affinity purification was generated by labeling the Rpn11 proteasome subunit with an HTBH polypeptide sequence that carries two sequences of six histidines (H), a TEV protease cleavage site (T), and a biotin-like sequence (B). The peptide binds strongly to avidin, which makes it possible to isolate proteasomes on an avidin carrier, followed by elution from the carrier using TEV protease cleavage [2,16].…”

“…A regulatory peptide, identified by a label, is bound to the ubiquitin chain by its fragment and is cleaved by the 26S proteasome. It is clear that a failure in its work will lead to a stop of the cell cycle at one or another phase [16].…”

Overexpression of Nfe2l1 increases proteasome activity and delays vision loss in a preclinical model of human blindness

“…An alternative approach to increase the proteolytic capacity of cells and treat human diseases caused by protein misfolding might include increasing the total proteasome pool. Several studies in diverse experimental systems, including mouse fibroblasts, the brain, the retina, and muscles, reported higher levels of proteasomes under genetic activation of the mTORC1 (mechanistic target of rapamycin complex 1) pathway (22)(23)(24)(25)(26). The mechanisms driving this transcriptional program have not been fully characterized but are proposed to be triggered by sterol-regulatory element binding protein 1 (Srebp1)-mediated transcriptional upregulation of the nuclear factor erythroid-2-like 1 (Nfe2l1) transcription factor (22,27).…”

Fluspirilene Analogs Activate the 20S Proteasome and Overcome Proteasome Impairment by Intrinsically Disordered Protein Oligomers

“…More recently, it has been recognized that the 20S proteasome plays a critical role in maintaining proteostasis by regulating autophagy and the direct degradation of oxidatively damaged and disordered proteins. ,,,− The 20S proteasome may therefore serve as the default protease to unremittently maintain low levels of IDPs, without the need for post-translational modifications, including protein ubiquitination. , Especially highly disordered proteins appear to be the main target of the 20S proteasome . Unfortunately, when dysregulation of IDPs production outpaces its clearance by the proteasome, the oligomeric forms in turn will inhibit the 20S proteasome, resulting in a downward spiral of IDP accumulation, oligomerization, and disease progression. ,,− These findings have sparked the search for small molecules capable of enhancing the proteolytic activity of the 20S proteasome. − ,− Small molecule proteasome enhancers are still relatively scarce, with only a few bona fide examples reported in the literature. − In this study, small molecule activation of the 20S proteasome was explored to enhance degradation of IDPs associated with these diseases and to overcome impairment of the 20S proteasome by pathogenic IDP oligomers.…”