The N-degron pathway mediates lipophagy: The chemical modulation of lipophagy in obesity and NAFLD.

BACKGROUND AND AIMS: Central to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) is the accumulation of lipids in the liver and various fat tissues. We aimed to elucidate the mechanisms by which lipid droplets (LDs) in the liver and adipocytes are degraded by the autophagy-lysosome system and develop therapeutic means to modulate lipophagy, i.e., autophagic degradation of LDs. METHODS: We monitored the process in which LDs are pinched off by autophagic membranes and degraded by lysosomal hydrolases in cultured cells and mice. The autophagic receptor p62/SQSTM-1/Sequestosome-1 was identified as a key regulator and used as a target to develop drugs to induce lipophagy. The efficacy of p62 agonists was validated in mice to treat hepatosteatosis and obesity. RESULTS: We found that the N-degron pathway modulates lipophagy. This autophagic degradation initiates when the molecular chaperones including BiP/GRP78, retro-translocated from the endoplasmic reticulum, is N-terminally (Nt-) arginylated by ATE1 R-transferase. The resulting Nt-arginine (Nt-Arg) binds the ZZ domain of p62 associated with LDs. Upon binding to Nt-Arg, p62 undergoes self-polymerization and recruits LC3+ phagophores to the site of lipophagy, leading to lysosomal degradation. Liver-specific Ate1 conditional knockout mice under high fat diet developed severe NAFLD. The Nt-Arg was modified into small molecule agonists to p62 that facilitate lipophagy in mice and exerted therapeutic efficacy in obesity and hepatosteatosis of wild-type but not p62 knockout mice. CONCLUSIONS: Our results show that the N-degron pathway modulates lipophagy and provide p62 as a drug target to treat NAFLD and other diseases related with metabolic syndrome.

The Cys-N-degron pathway modulates pexophagy through the N-terminal oxidation and arginylation of ACAD10.

In the N-degron pathway, N-recognins recognize cognate substrates for degradation via the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). We have recently shown that the autophagy receptor SQSTM1/p62 (sequestosome 1) is an N-recognin that binds the N-terminal arginine (Nt-Arg) as an N-degron to modulate autophagic proteolysis. Here, we show that the N-degron pathway mediates pexophagy, in which damaged peroxisomal fragments are degraded by autophagy under normal and oxidative stress conditions. This degradative process initiates when the Nt-Cys of ACAD10 (acyl-CoA dehydrogenase family, member 10), a receptor in pexophagy, is oxidized into Cys sulfinic (CysO2) or sulfonic acid (CysO3) by ADO (2-aminoethanethiol (cysteamine) dioxygenase). Under oxidative stress, the Nt-Cys of ACAD10 is chemically oxidized by reactive oxygen species (ROS). The oxidized Nt-Cys2 is arginylated by ATE1-encoded R-transferases, generating the RCOX N-degron. RCOX-ACAD10 marks the site of pexophagy via the interaction with PEX5 and binds the ZZ domain of SQSTM1/p62, recruiting LC3+-autophagic membranes. In mice, knockout of either Ate1 responsible for Nt-arginylation or Sqstm1/p62 leads to increased levels of peroxisomes. In the cells from patients with peroxisome biogenesis disorders (PBDs), characterized by peroxisomal loss due to uncontrolled pexophagy, inhibition of either ATE1 or SQSTM1/p62 was sufficient to recover the level of peroxisomes. Our results demonstrate that the Cys-N-degron pathway generates an N-degron that regulates the removal of damaged peroxisomal membranes along with their contents. We suggest that tannic acid, a commercially available drug on the market, has a potential to treat PBDs through its activity to inhibit ATE1 R-transferases.Abbreviations: ACAA1, acetyl-Coenzyme A acyltransferase 1; ACAD, acyl-Coenzyme A dehydrogenase; ADO, 2-aminoethanethiol (cysteamine) dioxygenase; ATE1, arginyltransferase 1; CDO1, cysteine dioxygenase type 1; ER, endoplasmic reticulum; LIR, LC3-interacting region; MOXD1, monooxygenase, DBH-like 1; NAC, N-acetyl-cysteine; Nt-Arg, N-terminal arginine; Nt-Cys, N-terminal cysteine; PB1, Phox and Bem1p; PBD, peroxisome biogenesis disorder; PCO, plant cysteine oxidase; PDI, protein disulfide isomerase; PTS, peroxisomal targeting signal; R-COX, Nt-Arg-CysOX; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNP, sodium nitroprusside; UBA, ubiquitin-associated; UPS, ubiquitinproteasome system.

Chemical modulation of SQSTM1/p62-mediated xenophagy that targets a broad range of pathogenic bacteria.

The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette-Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens.Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette-Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

TREM2 promotes natural killer cell development in CD3-CD122+NK1.1+ pNK cells.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer's disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells. RESULTS: Here, we demonstrated for the first time that CD3-CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice. CONCLUSIONS: Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

Cyclophilin A is an endogenous ligand for the triggering receptor expressed on myeloid cells-2 (TREM2).

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor expressed on macrophages, microglial cells, and pre-osteoclasts, and that participates in diverse cellular function, including inflammation, bone homeostasis, neurological development, and coagulation. In spite of the indispensable role of the TREM2 protein in the maintenance of immune homeostasis and osteoclast differentiation, the exact ligand for TREM2 has not yet been identified. Here, we report a putative TREM2 ligand which is secreted from MC38 cells and identified as a cyclophilin A (CypA). A specific interaction between CypA and TREM2 was shown at both protein and cellular levels. Exogenous CypA specifically interacted and co-localized with TREM2 in RAW264.7 cells, and the physical interactions were shown to regulate TREM2 signaling transduction. The Pro144 residue in the extracellular domain of TREM2 was found to be the specific binding site of CypA. When considered together, this provides evidence that CypA interacts specifically with TREM2 as a potent ligand.

Axl is a key regulator of intestinal γδ T-cell homeostasis.

Gut-homing γδ T cells are induced by chemokines and cell adhesion molecules and play a critical role in homeostasis and mucosal immunity; however, little is known regarding their upstream regulators. We investigated the role of Axl as a specific regulator of chemokines and cell adhesion molecule in the distribution of intestinal γδ T cells. The population of γδ T-cell receptor-positive cells including Vγ1 and Vγ7 subsets was remarkably increased in the intraepithelial lymphocytes of Axl-/- mice compared with those of wild-type (WT) mice. An increased number of migrated γδ T cells were observed in the coculture with intraepithelial cells from Axl-/- mice. The mRNA expression level of chemokine (C-C motif) ligand (CCL) 25 was specifically higher in the small intestine of Axl-/- mice than in WT mice. In adoptive transfer, the migration of both thymic and extrathymic γδ T cells was increased in Axl-/- mice. The activation of Axl signaling down-regulated CCL25 expression via ERK signaling pathway and reduced the population of γδ T cells. Systemic dissemination was suppressed in Axl-/- mice infected with Salmonella typhimurium. Thus, our findings suggest that Axl plays a critical role in regulating the migration of γδ T cells for the maintenance of homeostasis and bacterial resistance.-Kim, S.-M., Park, M., Yee, S.-M., Ji, K.-Y., Lee, E.-H., Nguyen, T.-V., Nguyen, T. H.-L., Jang, J., Kim, E.-M., Choi, H.-R., Yun, C.-H., Kang, H.-S. Axl is a key regulator of intestinal γδ T-cell homeostasis.

Daucosterol suppresses dextran sulfate sodium (DSS)-induced colitis in mice.

The effects of daucosterol have been identified in cancer therapy and neuronal diseases. However, the regulatory function of daucosterol in DSS-induced colitis has not yet been investigated. In this study, we evaluated the immunological and therapeutic effects of daucosterol in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Unlike vehicle mice, mice pre- or post-treated with daucosterol showed inhibition of body weight loss and the decrease in the disease activity index (DAI). In addition, daucosterol treatment rescued the DSS-induced decrease in colon length and disruption of the epithelial lining. Furthermore, it reduced DSS-induced production of reactive oxygen species (ROS), infiltration of macrophages, and expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß. Mice with colitis showed a decreased population of Foxp3+ cells, which was upregulated by daucosterol treatment. Furthermore, daucosterol increased natural killer (NK) cell activity and inhibited excessive IgA levels in mice with DSS-induced colitis. Collectively, our findings demonstrated that daucosterol significantly alleviated DSS-induced colitis, indicating the possibility of daucosterol as a therapeutic option for colitis.

The endoplasmic reticulum-residing chaperone BiP is short-lived and metabolized through N-terminal arginylation.

BiP and other endoplasmic reticulum (ER)-resident proteins are thought to be metabolically stable and to function primarily in the ER lumen. We sought to assess how the abundance of these proteins dynamically fluctuates in response to various stresses and how their subpopulations are relocated to non-ER compartments such as the cytosol. We showed that the molecular chaperone BiP (also known as GRP78) was short-lived under basal conditions and ER stress. The turnover of BiP was in part driven by its amino-terminal arginylation (Nt-arginylation) by the arginyltransferase ATE1, which generated an autophagic N-degron of the N-end rule pathway. ER stress elicited the formation of R-BiP, an effect that was increased when the proteasome was also inhibited. Nt-arginylation correlated with the cytosolic relocalization of BiP under the types of stress tested. The cytosolic relocalization of BiP did not require the functionality of the unfolded protein response or the Sec61- or Derlin1-containing translocon. A key inhibitor of the turnover and Nt-arginylation of BiP was HERP (homocysteine-responsive ER protein), a 43-kDa ER membrane-integrated protein that is an essential component of ER-associated protein degradation. Pharmacological inhibition of the ER-Golgi secretory pathway also suppressed R-BiP formation. Finally, we showed that cytosolic R-BiP induced by ER stress and proteasomal inhibition was routed to autophagic vacuoles and possibly additional metabolic fates. These results suggest that Nt-arginylation is a posttranslational modification that modulates the function, localization, and metabolic fate of ER-resident proteins.

TREM2 promotes Aß phagocytosis by upregulating C/EBPα-dependent CD36 expression in microglia.

TREM2 plays a critical role in the alleviation of Alzheimer's disease by promoting Aß phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic activity of Aß remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed Aß was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBPα, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBPα binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBPα was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer's disease by regulating C/EBPα-dependent CD36 expression and the consequent Aß phagocytosis.

Blockade of Axl signaling ameliorates HPV16E6-mediated tumorigenecity of cervical cancer.

Axl receptor tyrosine kinase is involved in the tumorigenesis and metastasis of many cancers. Axl expression was markedly higher in human papilloma virus type 16E6 (HPV16E6)-overexpressing HeLa (HE6F) cells and lower in HPV16E6-suppressing CaSki (CE6R) cells than in the controls. SiRNA-mediated knockdown of E6 expression led to increased phosphatase and tensin homolog (PTEN) phosphorylation at Ser380 and attenuated AKT phosphorylation. Expression of membrane-associated guanylate kinase inverted-2 (MAGI-2), an E6-induced degradation target, was induced in E6-siRNA-transfected cells. Moreover, myeloid zinc finger protein 1 (MZF1) binds directly to the Axl promoter in HE6F cells. Axl expression was regulated by HPV16E6-mediated PTEN/AKT signalling pathway, and Axl promoter activity was regulated through MZF1 activation in cervical cancer, which promoted malignancy. Axl silencing suppressed the metastasis of Caski cells and enhanced the susceptibility to NK cell-mediated killing of HE6F cells. In addition, the expression of Axl and MZF1 was highly correlated with clinical stage of cervical cancer and HPV16/18 infection. Taken together, Axl expression was induced by HPV16E6 in cervical cancer cells, suggesting that blockade of Axl signalling might be an effective way to reduce the progression of cervical cancer.

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma.

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma.

Axl signaling induces development of natural killer cells in vitro and in vivo.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axl antibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rß (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment of Axl-Ig. Anti-Axl antibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axl antibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83 % by the administration of mNK cells treated with anti-Axl antibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

Bacterial ß-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells.

Bacterial ß-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield ß-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial ß-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial ß-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial ß-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1ß, IL-6 and IL-17A/F, were markedly decreased in the colon of ß-(1,3)-glucan-pretreated mice. ß-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, ß-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial ß-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

Differential gene expression profiles in spontaneously hypertensive rats induced by administration of enalapril and nifedipine.

Enalapril and nifedipine are used as antihypertensive drugs; however, the therapeutic target molecules regulated by enalapril and nifedipine have yet to be fully identified. The aim of this study was to identify novel target genes that are specifically regulated by enalapril and nifedipine in tissues from spontaneously hypertensive rats (SHR) using DNA microarray analysis. We found that administration of SHR with enalapril and nifedipine differentially regulated 33 genes involved in the pathogenesis of cardiovascular diseases. Furthermore, we identified 16 genes that have not previously been implicated in cardiovascular diseases, including interleukin-24 (IL-24). Among them, exogenous administration of IL-24 attenuated the expression of vascular inflammation and hypertension-related genes induced by H2O2 treatment in mouse vascular smooth muscle (MOVAS) cells. This study provides valuable information for the development of novel antihypertensive drugs. In addition, the genes identified may be of use as biomarkers and therapeutic targets for cardiovascular diseases, including hypertension.

Interleukin-24 attenuates ß-glycerophosphate-induced calcification of vascular smooth muscle cells by inhibiting apoptosis, the expression of calcification and osteoblastic markers, and the Wnt/ß-catenin pathway.

Vascular calcification is a hallmark of cardiovascular disease. Interleukin-24 (IL-24) has been known to suppress tumor progression in a variety of human cancers. However, the role of IL-24 in the pathophysiology of diseases other than cancer is unclear. We investigated the role of IL-24 in vascular calcification. IL-24 was applied to a ß-glycerophosphate (ß-GP)-induced rat vascular smooth muscle cell (VSMC) calcification model. In this study, IL-24 significantly inhibited ß-GP-induced VSMC calcification, as determined by von Kossa staining and calcium content. The inhibitory effect of IL-24 on VSMC calcification was due to the suppression of ß-GP-induced apoptosis and expression of calcification and osteoblastic markers. In addition, IL-24 abrogated ß-GP-induced activation of the Wnt/ß-catenin pathway, which plays a key role in the pathogenesis of vascular calcification. The specificity of IL-24 for the inhibition of VSMC calcification was confirmed by using a neutralizing antibody to IL-24. Our results suggest that IL-24 inhibits ß-GP-induced VSMC calcification by inhibiting apoptosis, the expression of calcification and osteoblastic markers, and the Wnt/ ß-catenin pathway. Our study may provide a novel mechanism of action of IL-24 in cardiovascular disease and indicates that IL-24 is a potential therapeutic agent in VSMC calcification.