Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy

Tatiana A Giovannucci,Florian A Salomons,Henriette Stoy,Laura K Herzog,Shanshan Xu,Weixing Qian,Lara G Merino,Maria E Gierisch,Martin Haraldsson,Alf H Lystad,Hanna Uvell,Anne Simonsen,Anna-Lena Gustavsson,Michaela Vallin,Nico P Dantuma

doi:10.1080/15548627.2021.1988359

Tatiana A Giovannucci, Florian A Salomons + Show 13 more

Open Access

https://doi.org/10.1080/15548627.2021.1988359

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Abstract

ABSTRACT The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

Highlights

Intracellular protein homeostasis, i.e., proteostasis, depends on efficient recycling of defective proteins [1]
In subsequent analysis of the cellular response to CBK79, we found that administration of CBK79 to human osteosarcoma (HOS) cells expressing green fluorescent protein (GFP)tagged MAP1LC3B/LC3B caused the formation of cytosolic GFPLC3B foci, which is indicative for an effect on autophagy (Figure 3A)
Efficient and tightly regulated intracellular degrada tion is essential for the viability of practically all cells, the increased dependency of malignant cells on these proteolytic systems gives rise to a therapeutic window where curtailing their capacity is lethal for cancer cells without causing substantial harm to other cells [16]

Summary

Introduction

Intracellular protein homeostasis, i.e., proteostasis, depends on efficient recycling of defective proteins [1]. The ubiquitin-proteasome system (UPS) and macroautophagy ( referred to as autophagy) are the main proteolytic systems responsible for eliminating aberrant and misfolded proteins that are intercepted by protein quality control systems. The UPS is a complex system that involves a large number of proteins and is characterized by two key players: the small protein modifier ubiquitin, which tags proteins designated for degradation, and the proteasome, a multi-subunit proteolytic complex that executes the destruction of these proteins [2]. The compartmentalized nature of the proteasome requires proteins to be unfolded in order to be translocated into the proteolytic chamber. This requirement renders difficult-tountangle, aggregated proteins resistant to degradation by the UPS [3]. A failure in the timely degradation of misfolded proteins typically gives rise to the appearance of insoluble protein aggregates [4]

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