N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis

Abstract

The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D-CDC6 and D-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

N-degron and C-degron pathways of protein degradation

“…1G and SI Appendix, Figs. S2A and S3) (13,26,32,33,47,49,50). The Nt-Asn, -Gln, -Glu, and -Asp residues (as well as Nt-Cys, under some conditions) are destabilizing because of enzymatic deamidation of Nt-Asn and -Gln, and Nt-arginylation of Nt-Asp, -Glu, and (oxidized) -Cys ( Fig.…”

“…Arginylation, Autophagy, and the Arg/N-Degron Pathway. Kwon and colleagues (13,49,50) discovered that p62/Sqstm1 (called p62 below), a component of the autophagy-lysosome system, is also a non-E3 Arg/N-recognin that binds to cytosolic proteins that bear either Nt-Arg or specific hydrophobic Nt-residues. p62 mediates the capture of these proteins by autophagy and their subsequent destruction in lysosomes (refs.…”

“…Either a proteasome inhibitor or natural stresses can up-regulate the p62/autophagy branch of the Arg/N-degron pathway, termed the Arg/N-degron p62 pathway (SI Appendix, Fig. S4) (12,13,49,50).…”

p62/SQSTM1 – steering the cell through health and disease

“…Similarly, SQSTM1 forms disulfide bonds via cysteine 113 after recognition of N-terminally arginylated substrates by its ZZ domain (Cha-Molstad et al, 2018). These substrates of the so called N-end rule pathway are then degraded through autophagy in addition to their canonical proteasomal degradation (Cha-Molstad et al, 2017;Yoo et al, 2018).…”

Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

“…It is mediated by the arginyl transferase encoded within Ate1 gene (2). Ate1 is essential in most eukaryotic systems and is implicated in regulation of many physiological pathways including proteolysis (3,4), response to stress and heat shock (5)(6)(7), embryogenesis (8)(9)(10), regenerative processes (11)(12)(13), and aging (14,15). Ate1 has recently been identified as a master regulator affecting disease-associated pathways (16)(17)(18), and its knockout results in embryonic lethality and severe developmental defects in mice (9,10,19,20).…”