Current Research

Proteasomal N-degron pathway.

N-degron The proteasomal N-degron pathway is a Ub-dependent protein modification system where a class of E3s called N-recognins recognize type-1 (basic) and type-2 (bulky hydrophobic) destabilizing N-terminal residues of short-lived proteins as an essential component of degrons called N-degron. A functional N-degron is composed of a destabilizing N-terminal residue, an accessible internal lysine as the site of poly-Ub chain formation and a characteristic conformational feature required for ubiquitylation and degradation. As an effort to understand the physiological functions and the underlying molecular mechanisms of the N-degron pathway, we began biochemical and genetic dissection of this pathway using mouse model system. Studies by us and others for 15 years identified E3 components and related proteins (UBR1 through UBR7), E2 enzymes (HR6A and HR6B), pre-N-degron-modifying enzymes (ATE1 and NTAN1), and oxidizing cofactors, oxygen (O2) and nitric oxide (NO). Characterization of these proteins revealed a fairly complicated enzymatic cascade, in which an N-terminal modification cascade conditionally destabilizes an otherwise stabilizing pre-N-degron into a destabilizing N-degron, which is in turn recognized for ubiquitylation by E3s containing a common substrate recognition domain called the UBR box (Fig. 1). In mammalian N-degron pathway, N-terminal Asn and Gln are tertiary destabilizing residues that function through deamidation into the secondary destabilizing residues Asp and Glu by the N terminal amidohydrolases NTAN1 and NTAQ1, respectively. N-terminal Asp and Glu are secondary destabilizing residues that function through conjugation with Arg by ATE1 R transferase to create the primary destabilizing residue Arg. N-terminal arginylation requires Arg-tRNAArg as a source of Arg, definding a unique tRNA-dependent Ub pathway. N terminal Cys also is a tertiary destabilizing residue in that it can be conditionally destabilized by oxidation into Cys sulfinate (CysO2) or Cys sulfonate (CysO3), whose structure mimicks Asp. Oxidized Cys residues are arginylated by ATE1 R-transferase to create the primary destabilizing residue Arg. The oxidation of N-terminal Cys requires O2 and NO (or their derivatives) as cofactors, and hemin (Fe(3+)-heme) inhibits ATE1 through both proteasomal degradation of ATE1 and a redox mechanism involving the formation of intramolecular disulfide. The primary destabilizing residue Arg together with other destabilizing N-termini are bound by N-recognins for ubiquitylation and subsequent proteolysis by the 26S proteasome.

Our research focuses on the functions and mechanisms of the mammalian N-degron pathway, a subset of the ubiquitin system. The N-degron pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have shown that i) the NTAN1-mediated N-terminal deamidation is involved in socially conditioned behavior, ii) the ATE1-mediated N-terminal arginylation is essential for cardiovascular development, iii) the N-terminal oxidation, a novel protein modification, mediates a novel oxygen-dependent protein degradation which may function as an oxygen sensor, iv) UBR1 and UBR2 are functionally overlapping downstream Ub ligases of ATE1, v) UBR1 is involved in fat metabolism and muscle protein degradation, whereas UBR2 is essential for homologous chromosome and female viability, and vi) the cooperative activity of UBR1 and UBR2 underlies ATE1-mediated cardiovascular development, and other biological processes including central nervous development, mesenchymal cell proliferation, and chromosome stability.

We are further characterizing these mutant mice to understand the molecular mechanisms underlying the functions of the N-degron pathway. In addition, we recently developed a peptide-based pulldown assay for identifying proteins (including target substrates) that interact with the N-degron components. Using this assay coupled with mass spectrometric analysis, we identified a 600 kDa-protein termed p600 that binds to N-terminal destabilizing residues. It is to be determined whether ubiquitylation of the N-degron pathway is mediated by the UBR1-UBR2-p600. Another focus of our research is to develop inhibitors of the N-degron pathway. We have previously developed protein-based bivalent inhibitors of UBR1. We now are developing the peptide-based bivalent inhibitors, for therapeutic applications.

Role of N-terminal arginylation as a bimodal degron for proteasomal or autophagic proteolysis via the N-degron pathway.

N-terminal arginylation (Nt-Arginylation) is known to be generate a degron that induces substrate ubiquitination and proteasomal degradation via the N-degron pathway. Through recent study, our lab identified autophagic adaptor p62 as a new recognin of N-degron pathway. A set of ER-residing proteins are Nt-arginylated, including BiP/GRP78, calreticulin (CRT), and protein disulfide isomerase (PDI). Their Nt-arginylation is induced by cytosolic double-stranded DNA as an indication of an invading DNA-containing pathogen in innate immunity. Nt-arginylated BiP (R-BiP) accumulates in the cytosol, where its Nt-Arg binds the ZZ domain of p62. Based on this finding, we identified that Nt-arginylation function as a bimodal degron that modulates the crosstalk between the UPS and autophagy. The endoproteolytic cleavage products, CDC6 and BRCA1, are Nt-arginylated by R-transferase (ATE1) and recognized and degraded through UPS in normal condition. Under proteotoxicity, however, we showed that these proteins are stabilized and become the modulators of macroautophagy through the interaction of their Nt-Arg to the ZZ domain of p62/STQSM/Sequestosome-1, autophagic adaptor. In autophagic proteolysis, p62 is a general receptor of various cellular arginylated proteins (Yoo et al., 2018). We also verified the structural mechanism of autophagic N-recognin, p62, for selective recognition of Nt-Arg through the ZZ domain (Zhang et al., 2018).

Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-degron pathway.

In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62, and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons of the N-degron pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone BiP (GRP78). Upon N-degron pathwy interaction with the Nt-Arg of arginylated BiP (R-BiP), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatoray mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.

Harnessing the N-degron Pathway for Targeted Protein Degradation.

Despite advances in traditional small-molecule inhibitors and immunotherapy, the vast majority of the human proteome, including pathological proteins with gain-of-function toxicity, are still undruggable. Targeted protein degradation (TPD) of these targets via small chimeric degraders offers an attractive and alternative modality in drug development. Traditional heterobifunctional protein degraders, including proteolysis-targeting chimera (PROTAC), have mostly been limited to inducing the ubiquitination of the target substrate for its proteasomal degradation via the ubiquitin-proteasome system. To date, platform technology for targeted proteolysis based on alternative means, such as ubiquitin-independent selective autophagy, is yet to be fully developed. Our research interests and focus resulted in the synthesis and characterization of a first-in-class chimeric protein degrader termed AUTOTAC (autophagy-targeting chimera) comprising a warhead for target specificity and a p62-ZZ domain-binding moiety for p62-dependent autophagic delivery. AUTOTAC promoted both the autophagic degradation and functional silencing of target oncoproteins and aggregation-prone proteins. Our results provide a novel platform technology in targeted proteolysis through p62- and Arg/N-degron-dependent selective autophagy.

The N-terminal cysteine is a sensor of oxygen stress.

Our study demonstrated that the N-terminal cysteine residue (Nt-Cys) of Cys/N-degron substrates functions as a bimodal sensor for both O2 and oxidative stress. In normoxia, the Nt-Cys2 is enzymatically oxidized by cysteamine (2-aminoethanethiol) dioxygenase (ADO) and arginylated by ATE1-encoded R-transferases, generating the Arg-CysO2(H) (R-CO2) N-degron. The R-CO2 induces Lys48 (K48)-linked ubiquitination by the N-recognins UBR1 and UBR2, leading to proteasomal degradation. Under oxidative stress, however, the Nt-Cys2 is chemically oxidized by reactive oxygen species (ROS) to generate the R-CO3 N-degron via arginylation. The R-CO3 redirects proteolytic flux via reprograming of ubiquitin (Ub) code to K27-linked ubiquitination by the N-recognin UBR4 and K63-linked ubiquitination by a novel N-recognin KCMF1. The K27/K63 Ub chains are recognized by the autophagic receptor p62/SQSTM1/Sequestosome-1 for lysosomal degradation. We suggest a universal oxygen sensing system in which two different reactions converges into a single system: Cys/N-degron pathway. Based on suggested oxygen-stress sensing mechanism regarding cytosolic Nt-Cys proteins, we are also evaluating the mechanisms and functionalities about Nt-Cys substrates destined for extracellular transports. Furthermore, we are currently evaluating ubiquitin-mediated degradation of targeted substrates mediated by N-recognin E3 ligase using synthetic ligands.

The Arg/N-degron Pathway in Innate Immunity.

Recently, it is known that N-termini of ER chaperones are Arginylated by ATE1 under certain stress conditions such as long-term proteasomal inhibition and double-stranded DNA recognition. This suggested that ATE1 plays a role not only in regulation of protein stability but also non-proteolytic process. Sensing cytosolic DNA is the essential process in immune system. There are several cytosolic DNA sensors that regulates the expression of type I interferons and induce anti-viral response of host cells against DNA viruses. The history of researches about cytosolic nucleic acid sensors is just begun a few years ago. Although the key regulators of this cellular mechanism have been actively discovered, a lot of regulation factors have to be unveiled to explain the complex molecular mechanisms. Now, we are looking forward to reveal the relationship between arginylated ER chaperones and anti-viral signaling pathway in response to cytosolic DNA.

Role of the N-degron Pathway in ER-phagy and ER Homeostasis

The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Our research interests and focus show that the N-degron pathway mediates ER-phagy. This autophagic degradation of the ER initiates when the transmembrane E3 ligase TRIM13/RFP2 is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62/SQSTM1/Sequestosome-1, forming a complex, which in turn undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to p62 ZZ domain that enhance ER-phagy and ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.

Role of the N-degron pathway in Xenophagy

Xenophagy is a type of selective autophagy, targeting invading pathogens including bacteria, protozoans, and viruses as part of the host immune response. In the process of Xenophagy, intracellular pathogens that are either inside the cytosol or in pathogen-containing vacuoles are first tagged by poly-ubiquitin chains, following which they get recognized by the autophagy adaptor molecules such as p62, NBR1, NDP52, Optineurin and Tax1bp1. Recognized pathogens are targeted to a spherical structure with double layer membranes called autophagosome which fuses with lysosome to degrade intravesicular pathogens.

Role of the Cys N-degron pathway in Zellweger syndrome / Pexophagy

The peroxisome is a single lipid bilayer membrane structure that contains more than 50 hydrolytic enzymes. Main functions of peroxisome are beta-oxidative degradation of fatty acid. Number of peroxisome regulated by pexophagy, autophagic degradation of peroxisome. Recently, we found RCOX motif enhances peroxisomal targeting, plays as a pexophagy receptor, and accelerates pexophagy. Zellweger syndrome, one of the peroxisomal biogenesis disorders (PBDs), is caused by mutation of peroxisomal genes, PEXs which induces accelerated pexophagy. Zellweger syndrome patients show malfunction of body and life span of severe Zellweger syndrome patients limited to less than 1 year. However, treatment is not available, yet. We try development of drug for Zellweger syndrome based on the mechanism of RCOX motif of Cys N-degron pathway.

Role of N-degron pathway in obesity / Lipophagy

Obesity is a disease that results from a combination of various causes such as socio-environmental, genetic, psychiatric and endocrine factors. According to the World Health Organization (WHO), the worldwide obesity population has increased by double since the 1980s, and in the United States, about 34% of the total population in 2013 was classified as obese. In recent years, the obesity rates in Asian and African countries have conspicuously increased.

The drugs that have been known so far can cause side effects associated with metabolic problems such as nutritional imbalance and emotional changes such as lethargy and depression.

Excessive accumulation of lipid in tissues and blood is the main causes of diverse metabolic syndrome with body weight gain. Abdominal fat increased by a high fat diet affects the body fat and sugar metabolism, and increases triglyceride levels in the blood. Increasing triglyceride levels in the blood causes the accumulation of triglycerides in various tissues. Hepatic disease can be mentioned as a representative metabolic syndrome in which lipid accumulated in tissues is a direct cause of the disease. In order to prevent and treat hepatic diseases, it is the most effective method that effectively removes lipid accumulated in tissues. The main purpose of using drugs to prevent or treat obesity is not simple weight loss, but the goal should be to correct metabolic abnormalities due to accumulated surplus body fat, and ultimately, the focus should be on improving the prognosis of metabolic syndrome, such as diabetes and hepatic diseases, which may be caused by obesity. Therefore, in the case of anti-obesity drugs, the selective reduction of surplus fat should be the primary goal without changing the overall metabolism.

Lipophagy system is known to function to remove lipid droplets accumulated in cells and the lipid droplets accumulated in cells are removed by the action of lipophagy (Mark J. Czaja et al., 2009, Nature). Thereafter, studies on its mechanism and activation method are actively underway. Therefore, if the lipophagy process can be controlled by a technical method, it can be used as an effective therapeutic agent that can replace existing anti-obesity agents that prevent the lipid accumulation through regulation of feeding patterns and energy metabolism. Therefore, we have conducted intensive studies to develop a drug.