ABSTRACT
The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.
Key words: ATE1 R-transferase, N-end rule pathway, protein arginylation, protein quality control, proteolysis
Although eukaryotic proteins are synthesized with the initiator N-terminal Met, the cleavage of Nt-Met or internal residues generates a large proteome bearing N-terminal residues other than Nt-Met. One long standing question is whether these newly generated N-termini have physiological functions. The causative relationship between the N-termini and half-lives of proteins is defined by the N-end rule. There are several branches of the N-end rule pathway depending on the nature of degrons and N-recognins. In the classical pathway (Fig. 1A), type-1 (Nt-Arg, Nt-Lys, and Nt-His; positively charged) and type-2 (Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, and Nt-Ile; bulky hydrophobic) residues act as ligands, called N-ligands, to N-recognins. Amongst these, Nt-Arg can be exposed by endoproteolytic cleavage or generated through post-translational modifications (PTMs) of Nt-Asn, Nt-Gln (deamidation and Nt-arginylation), Nt-Cys (oxidation and Nt-arginylation), Nt-Asp, and Nt-Glu (Nt-arginylation). Among these PTMs, Nt-arginylation of Nt-Asp, Nt-Glu, and oxidized Nt-Cys is mediated by ATE1-encoded Arg-tRNA transferases (R-transferases; EC 2.3.2). In the UPS, Nt-Arg is bound by UBR boxes of the N-recognins, UBR1, UBR2, UBR4/p600, and/or UBR5. Upon binding to N-ligands, N-recognins promote polyubiquitination and proteasomal degradation of the substrates (Fig. 1A).
Figure 1.
The mammalian N-end rule pathway in the UPS and autophagy. (A) In the UPS, the tertiary destabilizing Nt-Asn and Nt-Gln are deamidated by NTAN1 and WDYHV1/NTAQ1 into Nt-Asp and Nt-Glu, respectively. The secondary destabilizing residues Nt-Asp and Nt-Glu are arginylated by ATE1-encoded Rtransferases. Nt-Cys is oxidized and subsequently arginylated by ATE1. C* denotes oxidized Nt-Cys, either Cys-sulfinic acid (CysO2[H]) or Cys-sulfonic acid (CysO3[H]). Nt-Arg and other type 1 and type 2 residues are directly bound by N-recognins characterized by the UBR box, such as UBR1, UBR2, UBR4, and UBR5. Upon binding to N-degrons, N-recognins promote polyubiquitination and processive degradation of the substrates through the proteasome, which generates short peptides. (B) In autophagic proteolysis, the formation of cytosolic misfolded proteins triggers Nt-arginylation and cytosolic relocalization of ER-residing chaperones. Among Nt-arginylated ER proteins, R-HSPA5 binds to the autophagic receptor SQSTM1 and is co-targeted to the phagophore for lysosomal degradation into amino acids. In this proteolytic system, Nt-Arg acts as a cis-acting degron for Nt-arginylated proteins themselves and a trans-acting degron for associated cargoes, such as cytosolic misfolded proteins.
Approximately one third of human proteins are targeted to the ER-Golgi secretory pathway. Upon translocation of nascent polypeptides into the ER lumen, their signal sequences are cleaved off, exposing new N-termini on mature proteins. We speculated that subpopulations of these proteins may be exposed to the cytosol during stresses and undergo Nt-arginylation and other Nt-PTMs. Bioinformatic analysis of 498 ER-targeted proteins showed that approximately 27% of ER proteins bear putative N-end rule substrates. Among these, 25 expose the arginylation acceptor Nt-Asp or Nt-Glu, including Ca++-associated folding factors, such as molecular chaperones (HSPA5, HSP90B1/GRP94, and CALR) and oxidoreductases (PDI and DNAJC10/ErdJ5). We raised antibodies specific to their Nt-arginylated forms and confirmed that HSPA5, CALR, and the PDI are Nt-arginylated (Fig. 2). Nt-arginylation of ER proteins is co-induced with autophagy as part of innate immunity to cytosolic double-stranded DNA (dsDNA), indicative of invading DNA-containing microbes, such as viruses and bacteria.
Figure 2.
Model for the modulation of SQSTM1 activity through Nt-arginylation of ER chaperones. The model illustrates that the formation of autophagic protein cargoes, such as cytosolic misfolded proteins, stimulates Nt-arginylation of ER-residing proteins. Following arginylation, R-HSPA5 and other Nt-arginylated ER proteins accumulate in the stressed cytosol. Cytosolic R-HSPA5 binds the ZZ domain of SQSTM1 and induces a conformational change of SQSTM1, exposing PB1 and LC3-interaction region (LIR). Upon binding, Nt-Arg induces the self-oligomerization and aggregation of SQSTM1 and the interaction of SQSTM1 with LC3 anchored on the phagophore membranes. This leads to the co-delivery of the R-HSPA5-SQSTM1 complex to the autophagosome and subsequent degradation by lysosomal hydrolases into amino acids. In parallel, R-HSPA5 acts as a molecular chaperone which binds cytosolic misfolded proteins, leading to the formation of cargo-R-HSPA5-SQSTM1 aggregates. Through this mechanism, R-HSPA5 plays a role in autophagic protein quality control of aggregation-prone misfolded proteins destined to be degraded by autophagy.
In protein quality control, soluble misfolded proteins are ubiquitinated and normally degraded through the proteasome. However, if Ub-conjugated substrates are not readily degraded by the UPS, for example, owing to their aggregation-prone nature, they are redirected to lysosomes via autophagosomes. These cargoes are collected by SQSTM1, which interacts with Ub chains of misfolded proteins and undergoes self-aggregation. Cargo-SQSTM1 aggregates are delivered to autophagy through SQSTM1 interaction with LC3 on phagophores, the precursor to autophagosomes. As Ub chains exist in substrates for both the UPS and autophagy, there have been intense debates on how cells sense and react to autophagic cargoes by modulating SQSTM1 activities. To determine the function of arginylated ER proteins, we examined the intracellular localization of R-HSPA5. Cell fractionation and immunostaining analyses showed that Nt-arginylated HSPA5 (R-HSPA5) proteins accumulate in the cytosol and are targeted to LC3-positive phagosomes via SQSTM1 (Fig. 2). Autophagic targeting of R-HSPA5 requires both its Nt-Arg and SQSTM1. In vitro binding assays showed that the Nt-Arg of R-HSPA5 binds SQSTM1 through an N-ligand-binding domain, called the ZZ motif. Autophagic flux assays showed that R-HSPA5 is sequestered within autophagosomes and degraded by lysosomal hydrolases. What happens to SQSTM1 when bound by Nt-Arg? Using in vitro aggregation and binding assays, we found that the binding of Nt-Arg promotes SQSTM1 self-polymerization as well as interaction with LC3. As illustrated in Figure 2, these results suggest that upon binding to Nt-Arg of R-HSPA5, SQSTM1 undergoes a conformational change, which exposes PB1 and LC3-interacting region (LIR), leading to accelerated self-polymerization and LC3 interaction on phagophore membranes.
Another important question is the identity of cytosolic cargoes of R-HSPA5. We found that one common signal that induced Nt-arginylation of ER proteins is the formation of misfolded proteins destined for autophagic degradation (Fig. 2). Following induction during proteasomal inhibition or in response to cytosolic dsDNA, R-HSPA5 colocalizes with puncta positive for Ub conjugates, SQSTM1, and LC3. R-HSPA5 interacts with the YFP-CL1 model substrate of spontaneous misfolding. ATE1-knockout abolishes the ability of SQSTM1 to form cytosolic puncta under proteasomal inhibition. Our results suggested that the cargoes of R-HSPA5 include misfolded proteins destined to be degraded by autophagy, and retrospectively explains why arginylation is coinduced with ubiquitination in response to cytosolic dsDNA (see Fig. 2).
The selectivity in proteolysis is mainly governed by the recognition and interaction between degrons and cognate recognins. In the UPS, PTM-generated degrons are recognized by E3 Ub ligases that promote polyubiquitination and proteasomal degradation. To date, the best characterized PTM-generated degrons in the UPS are N-degrons. In contrast, PTM-generated degrons have not been reported in autophagy. As summarized in Figure 1B, our results show that PTM-generated Nt-Arg acts as a cis-acting degron for R-HSPA5 and a trans-acting degron for its cargoes, such as misfolded proteins. In addition, Nt-Arg acts as an activating ligand to SQSTM1 and a delivery determinant to the phagophore. Given the multi-functionality of Nt-Arg in autophagic proteolysis, it is not surprising that Nt-Arg was adopted as a signaling mediator in the course of evolution by which the formation of autophagic protein cargoes is sensed and linked to SQSTM1 activation. What is special about ER chaperones? In contrast to cytosolic chaperones, such as HSP70 (a functional homolog of HSPA5), ER-targeted polypeptides naturally carry signal peptides whose cleavage can generate the acceptors for Nt-PTMs. Thus, the ER is an ideal reservoir of arginylation-compatible substrates that can be readily relocated to the cytosol in response to the formation of autophagic cargoes. It remains to be investigated how many ER residents and clients are Nt-arginylated and play a role outside the ER during cellular stress responses.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by the World Class Institute (WCI 2009-002) Program Bio & Medical Technology Development Program (NRF-2014M3A9B5073938) funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea (grant number: WCI 2009-002), KRIBB Research Initiative Program, NIH grant HL083365 (to Y.T.K. and Song Li), the Basic Science Research Programs of the NRF funded by the MSIP (NRF-2013R1A2A2A01014170 to Y.T.K.) and by the Ministry of Education (NRF-2013R1A1A2058983 to Y.D.Y), the Brain Korea 21 PLUS Program (to SNU), the SNU Nobel Laureates Invitation Program (to A.C.), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF) (to A.C.), and the Israel Science Foundation (ISF) (to A.C.). A.C. is an Israel Cancer Research Fund (ICRF) USA Professor