Characterization of the autophagic N-degron pathway and monitoring its chemical modulation for therapeutic...

Cellular homeostasis relies on the regulated degradation of unnecessary or harmful biomaterials including proteins. The N-degron pathway plays a central role in quality control at the crossroad of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome system (ALS). In this pathway, single N-terminal (Nt) residues of substrate proteins act as degradation determinants, called N-degrons. In the UPS, N-degrons are recognized and bound by their cognate N-recognins such as a family of E3 Ub ligases carrying the UBR box to induce substrate ubiquitination for proteasomal degradation. Our earlier studies identified an autophagic version of the N-degron pathway that works cooperatively with the UPS. In this process, N-degrons bind the N-recognin p62/SQSTM1 and modulate its activity as an autophagy receptor, leading to lysosomal destruction of UPS-resistant cargoes. We also developed chemical mimetics of N-degrons that exert efficacy to accelerate the degradation of excessive or harmful biomaterials such as cellular wastes and subcellular organelles. Here, we describe biochemical assays to characterize p62 as an autophagic N-recognin. We also delve into methodologies to assess therapeutic efficacy of chemical N-degrons in the degradation of lipid droplets (LDs) through lipophagy and the clearance of invading bacteria through xenophagy. These protocols can be used to identify new ZZ-type N-recognins in mammals and other species as well as under various pathophysiological conditions in both the UPS and ALS.