The N-Degron Pathway Mediates ER-phagy.

The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex 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 the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.

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

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system.

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711.

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2-C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Lipidomics reveals that acupuncture modulates the lipid metabolism and inflammatory interaction in a mouse model of depression.

Depression is a serious disease that has considerable impact on lipid metabolism and inflammatory responses. Recent studies have shown that leptin, which is well known as a mediator of energy homeostasis and is a cytokine in inflammatory response, plays an important role in depression. Acupuncture is widely used to treat depression; however, the underlying mechanisms and the effect of acupuncture on depression remain poorly understood. In this study, we utilized the chronic restraint stress (CRS) induced depression model and acupuncture treatment was performed at KI10, LR8, LU8, LR4 (AP) or non-acupoint (NP). Then, lipidomics was applied to investigate the effects of acupuncture on lipid metabolism and analyze leptin signals in the brain and changes of immune markers. Acupuncture treatment at AP improved depression-like behavior in an open-field test, forced swimming test, and marble burying test. Concurrently, CRS mice treated with AP acupuncture (CRS + AP) had significantly lower levels of aspartate aminotransaminase (AST, liver injury markers) and exhibited different lipid patterns in liver lipidomic profiles. In particular, triglycerides (TGs) contributed the change of lipid patterns. Compared to the CRS mice, TGs with relatively high degrees of unsaturated fatty acids increased in the CRS + AP mice, but did not change in CRS mice treated with NP acupuncture (CRS + NP). The levels of leptin in plasma and leptin receptor positive cells in the brain (hypothalamus and hippocampus) decreased and increased, respectively, in the CRS + AP mice, while opposite patterns were exhibited in the CRS and CRS + NP mice. These results indicated that acupuncture treatment at AP attenuated leptin insensitivity in CRS mice. Additionally, expression of pro-inflammatory cytokines such as interleukin-1 beta and tumor necrosis factor-alpha were decreased in the spleen, plasma, and liver of CRS + AP mice, which was one of results of alleviation of leptin resistance. In conclusion, these results show that AP acupuncture treatment effectively alleviated the depression-like behavior, affected immune responses, and altered hepatic lipid metabolism through the attenuation of leptin insensitivity.

Differential Pharmacological Activities of Oxygen Numbers on the Sulfoxide Moiety of Wasabi Compound 6-(Methylsulfinyl) Hexyl Isothiocyanate in Human Oral Cancer Cells.

6-(methylsulfinyl) hexyl isothiocyanate (6-MITC) is a naturally occurring compound isolated from Wasabia japonica (wasabi). The synthetic derivatives, 6-(methylsulfenyl) hexyl isothiocyanate (I7447) and 6-(methylsulfonyl) hexyl isothiocyanate (I7557), were derived from 6-MITC with the deletion and addition of oxygen, respectively. We aimed to evaluate the effect of these synthetic compounds on human oral cancer cells, SAS and OECM-1. All three compounds (I7447, 6-MITC, and I7557) inhibited the viability of SAS and OECM-1 cells using MTT assay. Morphological observations showed various proportions of mitotic arrest and apoptosis in cells treated with these compounds. Cell cycle analysis revealed relatively abundant G2/M arrest in 6-MITC and I7557-treated cells, whereas sub-G1 accumulation was found in I7447-treated cells. In using phosphorylated histone H3 as a marker for mitosis, the addition of 6-MITC and I7557 (excluding I7447) could be shown to arrest cells during mitosis. In contrast, I7447 induced more prominent apoptosis than the 6-MITC or I7557 compounds. The down-regulated expression of the phosphorylated form of CHK1 and Cdc25c was noted in 6-MITC and I7557-treated cells. I7557 could sensitize SAS cells to death by radiation. The wasabi compound, 6-MITC, and its chemical derivatives with different numbers of oxygen may have differential pharmacological effects on human oral cancer cells.

Effect of subcutaneous treatment with human umbilical cord blood-derived multipotent stem cells on peripheral neuropathic pain in rats.

In this study, we aim to determine the in vivo effect of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) on neuropathic pain, using three, principal peripheral neuropathic pain models. Four weeks after hUCB-MSC transplantation, we observed significant antinociceptive effect in hUCB-MSC-transplanted rats compared to that in the vehicle-treated control. Spinal cord cells positive for c-fos, CGRP, p-ERK, p-p 38, MMP-9 and MMP 2 were significantly decreased in only CCI model of hUCB-MSCs-grafted rats, while spinal cord cells positive for CGRP, p-ERK and MMP-2 significantly decreased in SNL model of hUCB-MSCs-grafted rats and spinal cord cells positive for CGRP and MMP-2 significantly decreased in SNI model of hUCB-MSCs-grafted rats, compared to the control 4 weeks or 8weeks after transplantation (p<0.05). However, cells positive for TIMP-2, an endogenous tissue inhibitor of MMP-2, were significantly increased in SNL and SNI models of hUCB-MSCs-grafted rats. Taken together, subcutaneous injection of hUCB-MSCs may have an antinociceptive effect via modulation of pain signaling during pain signal processing within the nervous system, especially for CCI model. Thus, subcutaneous administration of hUCB-MSCs might be beneficial for improving those patients suffering from neuropathic pain by decreasing neuropathic pain activation factors, while increasing neuropathic pain inhibition factor.

The Hot-Water Extract of Smilacis Chinae Rhizome Suppresses 2,4-Dinitrochlorobenzene and House Dust Mite-Induced Atopic Dermatitis-Like Skin Lesions in Mice.

Smilacis Chinae Rhizome (SCR) has been used as an oriental folk medicine for various biological activities. However, its effect on atopic dermatitis (AD) remains undetermined to date. We assessed the effect of orally administered hot-water extract of SCR on AD-like skin lesions in mice and its underlying mechanisms. AD-like murine model was prepared by repeated alternate application of house dust mite (Dermatophagoides farinae) extract (DFE) and 2,4-dinitrochlorobenzene (DNCB) for 4 weeks, topically to the ears. Daily oral administration of SCR for 3 and 4 weeks significantly reduced inflammatory ear thickening, with the effect being enhanced at the earlier start and longer period of administration. This effect was accompanied by a significant decrease in both Th2 and Th1 serum antibodies (total IgE, DFE-specific IgE, and IgG2a). Histological analysis showed that SCR markedly decreased the epidermal/dermal ear thickening and the dermal infiltration of inflammatory cells. Furthermore, SCR suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-17, IL-18, TSLP, and IFN-γ genes in the ear tissue. Taken together, our observations demonstrate that chronic oral administration of SCR exerts beneficial effect in mouse AD model, suggesting that SCR has the therapeutic potential as an orally active treatment of AD by modulating both Th1 and Th2 responses.

Experimental Mouse Models and Human Lung Organoid Models for Studying Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality throughout the world, is a highly complicated disease that includes chronic airway inflammation, airway remodeling, emphysema, and mucus hypersecretion. For respiratory function, an intact lung structure is required for efficient air flow through conducting airways and gas exchange in alveoli. Structural changes in small airways and inflammation are major features of COPD. At present, mechanisms involved in the genesis and development of COPD are poorly understood. Currently, there are no effective treatments for COPD. To develop better treatment strategies, it is necessary to study mechanisms of COPD using proper experimental models that can recapitulate distinctive features of human COPD. Therefore, this review will discuss representative established mouse models to investigate inflammatory processes and basic mechanisms of COPD. In addition, human COPD-mimicking human lung organoid models are introduced to help researchers overcome limits of mouse COPD models.

Ultrasound-triggered three dimensional hyaluronic acid hydrogel promotes in vitro and in vivo reprogramming into induced pluripotent stem cells.

Cellular reprogramming technologies have been developed with different physicochemical factors to improve the reprogramming efficiencies of induced pluripotent stem cells (iPSCs). Ultrasound is a clinically applied noncontact biophysical factor known for regulating various cellular behaviors but remains uninvestigated for cellular reprogramming. Here, we present a new reprogramming strategy using low-intensity ultrasound (LIUS) to improve cellular reprogramming of iPSCs in vitro and in vivo. Under 3D microenvironment conditions, increased LIUS stimulation shows enhanced cellular reprogramming of the iPSCs. The cellular reprogramming process facilitated by LIUS is accompanied by increased mesenchymal to epithelial transition and histone modification. LIUS stimulation transiently modulates the cytoskeletal rearrangement, along with increased membrane fluidity and mobility to increase HA/CD44 interactions. Furthermore, LIUS stimulation with HA hydrogel can be utilized in application of both human cells and in vivo environment, for enhanced reprogrammed cells into iPSCs. Thus, LIUS stimulation with a combinatorial 3D microenvironment system can improve cellular reprogramming in vitro and in vivo environments, which can be applied in various biomedical fields.

Structural insights into the distinct substrate preferences of two bacterial epoxide hydrolases.

Epoxide hydrolases (EHs), which catalyze the transformation of epoxides to diols, are present in many eukaryotic and prokaryotic organisms. They have recently drawn considerable attention from organic chemists owing to their application in the semisynthesis of enantiospecific diol compounds. Here, we report the crystal structures of BoEH from Bosea sp. PAMC 26642 and CaEH from Caballeronia sordidicola PAMC 26510 at 1.95 and 2.43 Å resolution, respectively. Structural analysis showed that the overall structures of BoEH and CaEH commonly possess typical α/ß hydrolase fold with the same ring-opening residues (Tyr-Tyr) and conserved catalytic triad residues (Asp-Asp-His). However, the two enzymes were found to have significantly different sequence compositions in the cap domain region, which is involved in the formation of the substrate-binding site in both enzymes. Enzyme activity assay results showed that BoEH had the strongest activity toward the linear aliphatic substrates, whereas CaEH had a higher preference for aromatic- and cycloaliphatic substrates. Computational docking simulations and tunnel identification revealed important residues with different substrate-binding preferences. Collectively, structure comparison studies, together with ligand docking simulation results, suggested that the differences in substrate-binding site residues were highly correlated with substrate specificity.

Enhancing Anti-Cancer Therapy with Selective Autophagy Inhibitors by Targeting Protective Autophagy.

Autophagy is a process of eliminating damaged or unnecessary proteins and organelles, thereby maintaining intracellular homeostasis. Deregulation of autophagy is associated with several diseases including cancer. Contradictory dual roles of autophagy have been well established in cancer. Cytoprotective mechanism of autophagy has been extensively investigated for overcoming resistance to cancer therapies including radiotherapy, targeted therapy, immunotherapy, and chemotherapy. Selective autophagy inhibitors that directly target autophagic process have been developed for cancer treatment. Efficacies of autophagy inhibitors have been tested in various pre-clinical cancer animal models. Combination therapies of autophagy inhibitors with chemotherapeutics are being evaluated in clinal trials. In this review, we will focus on genetical and pharmacological perturbations of autophagy-related proteins in different steps of autophagic process and their therapeutic benefits. We will also summarize combination therapies of autophagy inhibitors with chemotherapies and their outcomes in pre-clinical and clinical studies. Understanding of current knowledge of development, progress, and application of cytoprotective autophagy inhibitors in combination therapies will open new possibilities for overcoming drug resistance and improving clinical outcomes.

Monitoring the Activation of Selective Autophagy via N-Terminal Arginylation.

In addition to generating N-degron-carrying substrates destined for proteolysis, N-terminal arginylation can globally upregulate selective macroautophagy via activation of the autophagic N-recognin and archetypal autophagy cargo receptor p62/SQSTM1/sequestosome-1. To evaluate the macroautophagic turnover of cellular substrates, including protein aggregates (aggrephagy) and subcellular organelles (organellophagy) mediated by N-terminal arginylation in vivo, we report here a protocol for assaying the activation of the autophagic Arg/N-degron pathway and degradation of cellular cargoes via N-terminal arginylation. These methods, reagents, and conditions are applicable across a wide spectrum of different cell lines, primary cultures, and/or animal tissues, thereby providing a general means for identification and validation of putative cellular cargoes degraded by Nt-arginylation-activated selective autophagy.

Analyzing the Interaction of Arginylated Proteins and Nt-Arg-Mimicking Chemical Compounds to N-Recognins.

Characterizing and measuring the interactome of N-degrons and N-recognins are critical to the identification and verification of putative N-terminally arginylated native proteins and small-molecule chemicals that structurally and physiologically mimic the N-terminal arginine residue. This chapter focuses on in vitro and in vivo assays to confirm the putative interaction, and measure the binding affinity, between Nt-Arg-carrying natural (or Nt-Arg-mimicking synthetic) ligands and proteasomal or autophagic N-recognins carrying the UBR box or the ZZ domain. These methods, reagents, and conditions are applicable across a wide spectrum of different cell lines, primary cultures, and/or animal tissues, allowing for the qualitative analysis and quantitative measurement of the interaction of arginylated proteins and N-terminal arginine-mimicking chemical compounds to their respective N-recognins.

Biochemical and structural basis of mercuric reductase, GbsMerA, from Gelidibacter salicanalis PAMC21136.

Heavy metals, including mercury, are non-biodegradable and highly toxic to microorganisms even at low concentrations. Understanding the mechanisms underlying the environmental adaptability of microorganisms with Hg resistance holds promise for their use in Hg bioremediation. We characterized GbsMerA, a mercury reductase belonging to the mercury-resistant operon of Gelidibacter salicanalis PAMC21136, and found its maximum activity of 474.7 µmol/min/mg in reducing Hg+2. In the presence of Ag and Mn, the enzyme exhibited moderate activity as 236.5 µmol/min/mg and 69 µmol/min/mg, respectively. GbsMerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of GbsMerA and structural comparison with homologues indicated that GbsMerA contains residues, Tyr437´ and Asp47, which may be responsible for metal transfer at the si-face by providing a hydroxyl group (-OH) to abstract a proton from the thiol group of cysteine. The complex structure with NADPH indicated that Y174 in the re-face can change its side chain direction upon NADPH binding, indicating that Y174 may have a role as a gate for NADPH binding. Moreover, the heterologous host expressing GbsMerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking GbsMerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by GbsMerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.

Characterization and chemical modulation of p62/SQSTM1/Sequestosome-1 as an autophagic N-recognin.

In the Arg/N-degron pathway, single N-terminal (Nt) residues function as N-degrons recognized by UBR box-containing N-recognins that induce substrate ubiquitination and proteasomal degradation. Recent studies led to the discovery of the autophagic Arg/N-degron pathway, in which the autophagic receptor p62/SQSTM1/Sequestosome-1 acts as an N-recognin that binds the Nt-Arg and other destabilizing residues as N-degrons. Upon binding to Nt-Arg, p62 undergoes self-polymerization associated with its cargoes, accelerating the macroautophagic delivery of p62-cargo complexes to autophagosomes leading to degradation by lysosomal hydrolases. This autophagic mechanism is emerging as an important pathway that modulates the lysosomal degradation of various biomaterial ranging from protein aggregates and subcellular organelles to invading pathogens. Chemical mimics of the physiological N-degrons were developed to exert therapeutic efficacy in pathophysiological processes associated with neurodegeneration and other related diseases. Here, we describe the methods to monitor the activities of p62 in a dual role as an N-recognin and an autophagic receptor. The topic includes self-polymerization (for cargo condensation), its interaction with LC3 on autophagic membranes (for cargo targeting), and the degradation of p62-cargo complexes by lysosomal hydrolases. We also discuss the development and use of small molecule mimics of N-degrons that modulate p62-dependent macroautophagy in biological and pathophysiological processes.

Methods to detect AUTOphagy-Targeting Chimera (AUTOTAC)-mediated Targeted Protein Degradation in Tauopathies.

Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic. Graphical abstract Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper.

Mitophagy and endoplasmic reticulum-phagy accelerated by a p62 ZZ ligand alleviates paracetamol-induced hepatotoxicity.

BACKGROUND AND PURPOSE: Paracetamol (acetaminophen)-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. Autophagy is a degradative process by which various cargoes are collected by the autophagic receptors such as p62/SQSTM1/Sequestosome-1 for lysosomal degradation. Here, we investigated the protective role of p62-dependent autophagy in paracetamol-induced liver injury. EXPERIMENTAL APPROACH: Paracetamol-induced hepatotoxicity was induced by a single i.p. injection of paracetamol (500 mg·kg-1 ) in C57/BL6 male mice. YTK-2205 (20 mg·kg-1 ), a p62 agonist targeting ZZ domain, was co- or post-administered with paracetamol. Western blotting and immunocytochemistry were performed to explore the mechanism. KEY RESULTS: N-terminal arginylation of the molecular chaperone calreticulin retro-translocated from the endoplasmic reticulum (ER) was induced in the livers undergoing paracetamol-induced hepatotoxicity, and YTK-2205 exhibited notable therapeutic efficacy in acute hepatotoxicity as assessed by the levels of serum alanine aminotransferase and hepatic necrosis. This efficacy was significantly attributed to accelerated degradation of ubiquitin (Ub) conjugates as well as damaged mitochondria (mitophagy) and endoplasmic reticulum (ER-phagy). In primary murine hepatocytes treated with paracetamol, YTK-2205 induced the co-localization of p62+ LC3+ phagophores to the sites of mitophagy and ER-phagy. A similar activity of YTK-2205 was observed with N-acetyl-p-benzoquinone imine, a putative toxic metabolite of paracetamol in Hep3B cells. CONCLUSION AND IMPLICATIONS: Our results elucidated that p62-dependent autophagy plays a key role in the removal of cytotoxic materials such as damaged mitochondria in paracetamol-induced hepatotoxicity. Small molecule ligands to p62 may be developed into drugs to treat this pathological condition.