Purpose

The laboratory is interested in understanding the functions and mechanisms of ubiquitin (Ub)-dependent or autophagic proteolysis, with an emphasis on the N-degron pathway (N-end rule pathway). In Ubiquitin proteasome system (UPS), Ub is a 76 residue protein whose conjugation to other proteins regulates many biological processes including cell growth, division and differentiation; signal transduction; stress response; and thus the organismal processes as well, from embryonic development to immunity and functions of the nervous system. Ub-dependent proteolysis involves the marking of a target protein through covalent conjugation of Ub to an internal Lys residue of a substrate, which is mediated by the E1-E2-E3 enzymatic cascade. E1, the ATP-dependent Ub-activating enzyme, forms a high-energy thioester bond between the C-terminal Gly of Ub and a specific Cys of E1. The activated Ub is transesterified to a Cys residue of an E2 enzyme. Thereafter, a complex of E2 and E3 conjugates Ub to a Lys residue of a substrate protein. The functions of E3 include the recognition of a substrate' s degradation signal (degron). The ubiquitylated substrates are subsequently degraded by the 26S proteasome.

Autophagy is divided into macroautophagy and selective autophagy. Macroautophagy generates energy and amino acids through bulk degradation non-selectively during nutrient deprivation. Long-lived proteins and organelles are sequestered by autophagosomes and digested by lysosomal Hydrolases (Feng et al., 2014; Kaur and Debnath, 2015). However selective autophagy targets autophagy cargoes via specific receptors such as p62, NBR1 et al. Ubiquitin-associated (UBA)- and LC3-interating region(LIR) domains on these receptors enable recognition of both ubiquitin (Ub) chains on protein cargoes and LC3 on autophagosomes (Deng et al., 2017; Ji and Kwon, 2017). From misfolded proteins and their aggregates to organelles and invading pathogens would be targets of selective autophagy for quality control.

The N-degron pathway, had known as a subset of the Ub system, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Since 1994, we have identified/characterized mammalian genes encoding proteins that are directly involved in this proteolytic pathway, including N-terminal asparagine-specific amidase NTAN1, arginyl tRNA protein transferase (ATE1), ubiquitin ligases UBR1-UBR7, and ubiquitin conjugating enzymes HR6A and RAD6 (Kwon et al., 1998; 1999a, 2000; 2001; 2002; 2003; Tasaki et al., 2005). The N-degron pathway is mediated by an enzymatic cascade: deamidation, oxidation, arginylation, and ubiquitylation. In mammals, N-terminal Asn and Gln function as pre-N-degrons for the Ub system through their deamidation into Asp and Glu, respectively, by NTAN1 and NTAQ1 (Kwon et al., 2000). N-terminal Asp and Glu can be arginylated by ATE1-dependent N-terminal arginylation (Kwon et al., 1999a; 2002), a universal eukaryotic modification originally observed ~40 years ago. In addition to N-terminal Asp and Glu, N-terminal Cys also acts as pre-N-degron by its oxidation into Cys sulfinic acid (CysO2) or cysteic acid (CysO3), and subsequent arginylation by ATE1 (Lee et al., 2005). N-terminal Arg together with other primary destabilizing residues are recognized by special Ub ligases called N-recognins, including UBR1, UBR2, UBR4, and UBR5 (Kwon et al., 1998; 2001; Tasaki et al., 2005).

Recently our lab finds out that p62, adaptor of autophagy, roles as an N-recognin whose ZZ domain binding with a N-terminal arginine (Cha-molstad et al., 2015; 2017). Via interaction with p62, N-terminal arginine distributes substrates to autophagy as well as UPS and functions as a bimodal degron (Yoo et al., 2018). Through these functions of N-terminal arginine, N-degron pathway mediates crosstalk between UPS and autophagy. In normal conditions, the Nt-Arg of arginylated substrates is recognized by the UBR box of UBR proteins for degradation through the UPS. However, if proteolytic flux via the UPS is compromised, the Nt-Arg of the substrates is preferentially recognized by the ZZ domain of p62, facilitating p62 self-polymerization, autophagosome biogenesis, and lysosomal degradation of autophagic protein cargoes. Moreover, with roles of N-terminal arginine in autophagy, the N-degron pathway mediates the selective autophagy like reticulophagy (Ji et al., 2019), pexophagy, lipophagy, xenophagy et al. With these principles, N-terminal arginine mimic chemicals can facilitate degradation of misfolded proteins via mediating UPS or autophagy.

With principles that N-terminal arginine mimic chemicals bind to and activate p62 and autophagy, our lab studied about targeted degradation. Compounds comprising a specific warhead of target and a p62-ZZ domain-binding moiety which mediates p62-dependent autophagic delivery are synthesized and used for research of removing various proteins, aggregates and organelles causing diseases. Overall, in this laboratory, students and researchers are conducting broad spectrum researches based on the N-degron pathway.

The followings are some of our research interests.