ACOD1, rather than itaconate, facilitates p62-mediated activation of Nrf2 in microglia post spinal cord contusion.

BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.

ATF3/EGR1 regulates myocardial ischemia/reperfusion injury induced autophagy and inflammation in cardiomyocytes.

Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

Hyperacetylated microtubules assist porcine deltacoronavirus nsp8 to degrade MDA5 via SQSTM1/p62-dependent selective autophagy.

The microtubule (MT) is a highly dynamic polymer that functions in various cellular processes through MT hyperacetylation. Thus, many viruses have evolved mechanisms to hijack the MT network of the cytoskeleton to allow intracellular replication of viral genomic material. Coronavirus non-structural protein 8 (nsp8), a component of the viral replication transcriptional complex, is essential for viral survival. Here, we found that nsp8 of porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with a zoonotic potential, inhibits interferon (IFN)-ß production by targeting melanoma differentiation gene 5 (MDA5), the main pattern recognition receptor for coronaviruses in the cytoplasm. Mechanistically, PDCoV nsp8 interacted with MDA5 and induced autophagy to degrade MDA5 in wild-type cells, but not in autophagy-related (ATG)5 or ATG7 knockout cells. Further screening for autophagic degradation receptors revealed that nsp8 interacts with sequestosome 1/p62 and promotes p62-mediated selective autophagy to degrade MDA5. Importantly, PDCoV nsp8 induced hyperacetylation of MTs, which in turn triggered selective autophagic degradation of MDA5 and subsequent inhibition of IFN-ß production. Overall, our study uncovers a novel mechanism employed by PDCoV nsp8 to evade host innate immune defenses. These findings offer new insights into the interplay among viruses, IFNs, and MTs, providing a promising target to develop anti-viral drugs against PDCoV.IMPORTANCECoronavirus nsp8, a component of the viral replication transcriptional complex, is well conserved and plays a crucial role in viral replication. Exploration of the role mechanism of nsp8 is conducive to the understanding of viral pathogenesis and development of anti-viral strategies against coronavirus. Here, we found that nsp8 of PDCoV, an emerging enteropathogenic coronavirus with a zoonotic potential, is an interferon antagonist. Further studies showed that PDCoV nsp8 interacted with MDA5 and sequestosome 1/p62, promoting p62-mediated selective autophagy to degrade MDA5. We further found that PDCoV nsp8 could induce hyperacetylation of MT, therefore triggering selective autophagic degradation of MDA5 and inhibiting IFN-ß production. These findings reveal a novel immune evasion strategy used by PDCoV nsp8 and provide insights into potential therapeutic interventions.

Endothelium-specific deletion of p62 causes organ fibrosis and cardiac dysfunction.

BACKGROUND: The autophagy adapter SQSTM1/p62 is crucial for maintaining homeostasis in various organs and cells due to its protein-protein interaction domains and involvement in diverse physiological and pathological processes. Vascular endothelium cells play a unique role in vascular biology and contribute to vascular health. METHODS: Using the Cre-loxP system, we generated mice with endothelium cell-specific knockout of p62 mediated by Tek (Tek receptor tyrosine kinase)-cre to investigate the essential role of p62 in the endothelium. In vitro, we employed protein mass spectrometry and IPA to identify differentially expressed proteins upon knockdown of p62. Immunoprecipitation assays were conducted to demonstrate the interaction between p62 and FN1 or LAMC2 in human umbilical vein endothelium cells (HUVECs). Additionally, we identified the degradation pathway of FN1 and LAMC2 using the autophagy inhibitor 3-methyladenine (3-MA) or proteasome inhibitor MG132. Finally, the results of immunoprecipitation demonstrated that the interaction between p62 and LAMC2 was abolished in the PB1 truncation group of p62, while the interaction between p62 and FN1 was abolished in the UBA truncation group of p62. RESULTS: Our findings revealed that p62 Endo mice exhibited heart, lung, and kidney fibrosis compared to littermate controls, accompanied by severe cardiac dysfunction. Immunoprecipitation assays provided evidence of p62 acting as an autophagy adapter in the autophagy-lysosome pathway for FN1 and LAMC2 degradation respectively through PB1 and UBA domain with these proteins rather than proteasome system. CONCLUSIONS: Our study demonstrates that defects in p62 within endothelium cells induce multi-organ fibrosis and cardiac dysfunction in mice. Our findings indicate that FN1 and LAMC2, as markers of (EndoMT), have detrimental effects on HUVECs and elucidate the autophagy-lysosome degradation mechanism of FN1 and LAMC2.

Nuclear-cytoplasmic translocation of SQSTM1/p62 protein enhances ESCC cell migration and invasion by stabilizing EPLIN expression.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant disease with a poor prognosis. We previously found that p62 presented a marked nuclear-cytoplasmic translocation in ESCC cells as compared that in normal esophageal epithelial cells, but its effects on ESCC cells remain unclear. This study aims to clarify the impacts of different cellular localization of p62 on the function of ESCC cells and the underlying molecular mechanisms. We here demonstrated that cytoplasmic p62 enhances the migration and invasion abilities of esophageal cancer cells, whereas nuclear p62 has no effect. We further explored the interaction protein of p62 by using GST pull-down experiment and identified EPLIN as a potential protein interacting with p62. In addition, reducing EPLIN expression significantly inhibited the migration and invasion of ESCC cells, which were rescued when EPLIN expression was restored after the p62 knockdown. At a molecular level, p62 in cytoplasm positively regulated the expression of EPLIN via enhancing its protein stability. Data from the TCGA and GEO database displayed a significant up-regulation of EPLIN mRNA expression in ESCC tissues compared with corresponding paired esophageal epithelial samples. Our findings present evidence that the nuclear-cytoplasmic translocation of p62 protein contributes to an aggressive malignancy phenotype, providing candidate molecular biomarkers and potential molecular targets for the diagnosis and treatment of ESCC.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-ß1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-ß1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-ß1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-ß1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.

Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle.

SQSTM1/p62 bodies are phase-separated condensates that play a fundamental role in intracellular quality control and stress responses. Despite extensive studies investigating the mechanism of formation and degradation of SQSTM1/p62 bodies, the constituents of SQSTM1/p62 bodies remain elusive. We recently developed a purification method for intracellular SQSTM1/p62 bodies using a cell sorter and identified their constituents by mass spectrometry. Combined with mass spectrometry of tissues from selective autophagy-deficient mice, we identified vault, a ubiquitous non-membranous organelle composed of proteins and non-coding RNA, as a novel substrate for selective autophagy. Vault directly binds to NBR1, an SQSTM1/p62 binding partner recruited to SQSTM1/p62 bodies, and is subsequently degraded by selective autophagy dependent on the phase separation of SQSTM1/p62. We named this process "vault-phagy" and found that defects in vault-phagy are related to nonalcoholic steatohepatitis (NASH)-derived hepatocellular carcinoma. Our method for purifying SQSTM1/p62 bodies will contribute to elucidating the mechanisms of several stress responses and diseases mediated by SQSTM1/p62 bodies.

SQSTM1/p62 inhibition impairs pro-survival signaling in hypoxic human dendritic cells.

The sequestosome 1 (SQSTM1)/p62 is an adaptor protein which plays multiple roles in several cell functions, including cell survival and autophagy. Dendritic cells (DCs) are the most prominent antigen presenting cells and during their lifespan they are exposed to different oxygen tensions, including hypoxia. By using a siRNA approach we found out that p62 was implicated in the maintenance of Erk1/2 phosphorylation and preservation of hypoxic DC survival, as well as in the reduction of AMPK activation. Thus, p62 expression in DCs in hypoxic microenvironments, such as in the lymphoid organs, may extend their lifespan to ensure their functions.

Ovatodiolide induces autophagy-mediated cell death through the p62-Keap1-Nrf2 signaling pathway in chronic myeloid leukemia cells.

Ovatodiolide is a macrocyclic diterpenoid compound with various biological activities that displays considerable anticancer potential in different tumor models. However, the underlying mechanism for this antineoplastic activity remains unclear. The aim of the present study was to investigate the anticancer effect and possible molecular mechanism of ovatodiolide in human chronic myeloid leukemia (CML). Ovatodiolide suppressed cell colony formation and induced apoptosis in the K562 and KU812 cells. We also observed that ovatodiolide enhanced the production of reactive oxygen species (ROS), activated Nrf2 signaling, and inhibited mTOR phosphorylation. Autophagic flux was shown to be enhanced after treatment with ovatodiolide in K562 cells. Furthermore, autophagy inhibition alleviated ovatodiolide-induced cell apoptosis, whereas autophagy promotion aggravated apoptosis in CML cells. These results demonstrated that ovatodiolide activates autophagy-mediated cell death in CML cells. Additionally, ovatodiolide transcriptionally activated the expression of p62, and the p62 levels were negatively regulated by autophagy. Moreover, p62-Keap1-Nrf2 signaling was confirmed to be involved in ovatodiolide-induced cell death. Accordingly, LC3B knockdown augmented the ovatodiolide-induced p62 expression, increased the p62-Keap1 interaction, and enhanced the translocation of Nrf2 into the nucleus. In contrast, p62 inhibition abolished the effects that were induced through ovatodiolide treatment. Nrf2 inhibition with ML385 diminished the protective effect of autophagy inhibition in CML cells. Collectively, our results indicate that ovatodiolide induces oxidative stress and provokes autophagy, which effectively decreases the expression of p62 and weakens the protective effect of Nrf2 signaling activation, thus contributing to apoptosis in CML cells.

The PB1 and the ZZ domain of the autophagy receptor p62/SQSTM1 regulate the interaction of p62/SQSTM1 with the autophagosome protein LC3B.

Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. In this study, we investigated if the interaction of p62/SQSTM1 with LC3B could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3B. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3B. We identified a mechanism of regulation of p62/SQSTM1 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3B. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3B. Also, a small compound that binds to the ZZ domain enhances interaction with LC3B. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3B through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies.

Autophagy and nuclear morphometry are associated with histopathologic features in esophageal squamous cell carcinoma.

Less than 15% of patients with esophageal squamous cell carcinoma (ESCC) survive 5 years after diagnosis. A better understanding of the biology of these tumors and the development of clinical biomarkers is needed. Autophagy is a physiological mechanism involved in the turnover of cellular components that plays a key role in cancer. This study evaluated the differential levels of three key regulators of autophagy (SQSTM1, MAP1LC3B, and BECN1) in patients with ESCC, associating autophagy with histopathologic features, including the grade of differentiation, mitotic rate, inflammation score, and the intensity of tumor-infiltrating lymphocytes. Nuclear morphometry of the tumor parenchyma was also assessed, associating it with autophagy and histopathology. All three markers significantly increased in patients with ESCC compared to the control group. Based on the mean expression of each protein in the control group, 57% of patients with ESCC had high levels of all three markers compared to control patients (14%). The most frequent profiles found in ESCC were BECNhigh/MAP1LC3high and BECNhigh/SQSTM1high. According to the TCGA database, we found that the main autophagy genes were upregulated in ESCC. Moreover, high levels of autophagy markers were associated with a poor prognosis. Considering nuclear morphometry, ESCC samples showed a significant reduction in nuclear area, which was strongly negatively correlated with autophagy. Finally, the percentage of normal nuclei was associated with tumor differentiation, while poorly differentiated tumors showed lower SQSTM1 levels. ESCC progression may involve increased autophagy and changes in nuclear structure, associated with clinically relevant histopathological features. KEY MESSAGES: Autophagy markers are co-increased in primary ESCC. Autophagy negatively correlates with nuclear morphometry in ESCC parenchyma. Autophagy and nuclear morphometry are associated with histopathological features. Autophagy is increased in ESCC-TCGA database and associated with poor prognosis.

Pristimerin suppresses AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy.

Macroautophagy/autophagy plays an important role in regulating cellular homeostasis and influences the pathogenesis of degenerative diseases. Tendinopathy is characterized by tendon degeneration and inflammation. However, little is known about the role of selective autophagy in tendinopathy. Here, we find that pristimerin (PM), a quinone methide triterpenoid, is more effective in treating tendinopathy than the first-line drug indomethacin. PM inhibits the AIM2 inflammasome and alleviates inflammation during tendinopathy by promoting the autophagic degradation of AIM2 through a PYCARD/ASC-dependent manner. A mechanistic study shows that PM enhances the K63-linked ubiquitin chains of PYCARD/ASC at K158/161, which serves as a recognition signal for SQSTM1/p62-mediated autophagic degradation of the AIM2-PYCARD/ASC complex. We further identify that PM binds the Cys53 site of deubiquitinase USP50 through the Michael-acceptor and blocks the binding of USP50 to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains of PYCARD/ASC. Finally, PM treatment in vivo generates an effect comparable to inflammasome deficiency in alleviating tendinopathy. Taken together, these findings demonstrate that PM alleviates the progression of tendinopathy by modulating AIM2-PYCARD/ASC stability via SQSTM1/p62-mediated selective autophagic degradation, thus providing a promising autophagy-based therapeutic for tendinopathy.Abbreviations: 3-MA: 3-methyladenine; AIM2: absent in melanoma 2; AT: Achilles tenotomy; ATP: adenosine triphosphate; BMDMs: bone marrow-derived macrophages; CHX: cycloheximide; Col3a1: collagen, type III, alpha 1; CQ: chloroquine; Cys: cysteine; DARTS: drug affinity responsive target stability; DTT: dithiothreitol; DUB: deubiquitinase; gDNA: genomic DNA; GSH: glutathione; His: histidine; IL1B/IL-1ß: interleukin 1 beta; IND: indomethacin; IP: immunoprecipitation; LPS: lipopolysaccharide; MMP: mitochondrial membrane potential; NLRP3: NLR family, pyrin domain containing 3; PM: pristimerin; PYCARD/ASC: PYD and CARD domain containing; SN: supernatants; SOX9: SRY (sex determining region Y)-box 9; SQSTM1: sequestosome 1; Tgfb: transforming growth factor, beta; TIMP3: tissue inhibitor of metalloproteinase 3; TNMD: tenomodulin; TRAF6: TNF receptor-associated factor 6; Ub: ubiquitin; USP50: ubiquitin specific peptidase 50; WCL: whole cell lysates.

Effect of Nano-selenium on Biological Mechanism of Goblet Cells of the Small Intestine Within Laying Hen.

Dietary selenium intake within the normal physiological range is critical for various supporting biological functions. However, the effect of nano-selenium on biological mechanism of goblet cells associated with autophagy is largely unknown.The purpose of this study was to investigate the effect of nano-selenium on the mucosal immune-defense mechanism of goblet cells (GCs) in the small intestine of laying hens.The autophagy was determined by using specific markers. Nano-selenium-treated group of immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB) results indicated the strong positive immune signaling of microtubule-associated light chain (LC3) within the mucosal surface of the small intestine. However, weak expression of LC3 was observed in the 3-methyladenine autophagy inhibitor (3-MA) group. IHC and IF staining results showed the opposite tendency for LC3 of sequestosome 1 (P62/SQSTM1). P62/SQSTM1 showed strong positive immune signaling within the mucosal surface of the small intestine of the 3-MAgroup, and weak immune signaling of P62/SQSTM1 in the nano-selenium-treated group. Moreover, pinpointing autophagy was involved in the mucosal production and enrichment of mucosal immunity of the GCs. The morphology and ultrastructure evidence showed that the mucus secretion of GCs was significantly increased after nano-selenium treatment confirmed by light and transmission electron microscopy. Besides that, immunostaining of IHC, IF and WB showed that autophagy enhanced the secretion of Mucin2 (Muc2) protein in nano-selenium-treated group. This work illustrates that the nano-selenium particle might enhance the mucosal immune-defense mechanism via the protective role of GCs for intestinal homeostasis through autophagy.

KPT330 promotes the sensitivity of glioblastoma to olaparib by retaining SQSTM1 in the nucleus and disrupting lysosomal function.

ABBREVIATIONS: AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs: double-strand breaks; GBM: glioblastoma; HR: homologous recombination; H2AX: H2A.X variant histone; IHC: immunohistochemistry; LAPTM4B: lysosomal protein transmembrane 4 beta; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PARP: poly(ADP-ribose) polymerase; RAD51: RAD51 recombinase; SQSTM1: sequestosome 1; SSBs: single-strand breaks; RNF168: ring finger protein 168; XPO1: exportin 1.

PA2G4/EBP1 ubiquitination by PRKN/PARKIN promotes mitophagy protecting neuron death in cerebral ischemia.

Cerebral ischemia induces massive mitochondrial damage, leading to neuronal death. The elimination of damaged mitochondria via mitophagy is critical for neuroprotection. Here we show that the level of PA2G4/EBP1 (proliferation-associated 2G4) was notably increased early during transient middle cerebral artery occlusion and prevented neuronal death by eliciting cerebral ischemia-reperfusion (IR)-induced mitophagy. Neuron-specific knockout of Pa2g4 increased infarct volume and aggravated neuron loss with impaired mitophagy and was rescued by introduction of adeno-associated virus serotype 2 expressing PA2G4/EBP1. We determined that PA2G4/EBP1 is ubiquitinated on lysine 376 by PRKN/PARKIN on the damaged mitochondria and interacts with receptor protein SQSTM1/p62 for mitophagy induction. Thus, our study suggests that PA2G4/EBP1 ubiquitination following cerebral IR-injury promotes mitophagy induction, which may be implicated in neuroprotection.Abbreviations: AAV: adeno-associated virus; ACTB: actin beta; BNIP3L/NIX: BCL2 interacting protein 3 like; CA1: Cornu Ammonis 1; CASP3: caspase 3; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; PA2G4/EBP1: proliferation-associated 2G4; FUNDC1: FUN14 domain containing 1; IB: immunoblotting; ICC: immunocytochemistry; IHC: immunohistochemistry; IP: immunoprecipitation; MCAO: middle cerebral artery occlusion; MEF: mouse embryonic fibroblast; OGD: oxygen-glucose deprivation; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced kinase 1; RBFOX3/NeuN: RNA binding fox-1 homolog 3; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I; WT: wild-type.

Over-expression of microRNA-145 Elevating Autophagy Activities via Downregulating FRS2 Expression.

OBJECTIVES: Osteoarthritis (OA) is one of the most common chronic and progressive joint diseases characterized by cartilage degeneration and chondrocyte death. In this study, we aimed to identify the modulation effect of miR-145 on chondrocytes' autophagy during the development of OA. BACKGROUND: Osteoarthritis (OA) is one of the most prevalent types of chronic and progressive joint disorder with the symptoms of joint pain and stiffness, and it leads to disability at the end stage. In recent years, microRNA-145 (miR-145) has been found to activate autophagy in various cell types, including mesenchymal stem cells, cardiomyocytes, and osteosarcoma cells. However, it is unknown whether miR-145 regulates the progression of OA by influencing chondrocyte autophagy. METHODS: Before investigating the regulatory effect of miR-145 on the autophagic activity of chondrocytes, the expression of miR-145 in human joint samples was analyzed. The targeting relationship between miR-145 and FRS2 was detected by dual luciferase assay. The effect of FRS2 and miR-145 on the autophagic activity of chondrocytes was observed by bidirectional expression of FRS2 and miR-145. RESULTS: The miR-145 expression and LC3-II/LC3-I ratio were significantly decreased and the SQSTM1 expression was increased in OA patients. The miR-145 overexpression in C20A4 cells increased LC3-II/LC3-I ratio, decreased SQSTM1 expression, and was positively correlated with autophagic activity. Under oxidative stress, miR-145 overexpression significantly improved chondrocyte viability through autophagy stimulation. FRS2 is a potential target of miR-145 via a binding sequence within its 3' UTR. FRS2 acts as the downstream mediator of miR-145 to suppress autophagy through activating PI3K/Akt/mTOR pathways. CONCLUSION: The miR-145 acts as a protective factor against chondrocytes by regulating miRFRS2- autophagy axis. The decrease of miR-145 in articular synovial fluid may turn out to be an important marker for early diagnosis of OA, and modulation of miR-145 may represent a promising therapeutic strategy for OA.

N-acetylneuraminic acid modulates SQSTM1/p62 sialyation-mediated ubiquitination degradation contributing to vascular endothelium dysfunction in experimental atherosclerosis mice.

Sialic acid (SIA) has been reported to be a risk factor for atherosclerosis (AS) due to its high plasma levels in such patients. However, the effect of increasing SIA in circulation on endothelial function during AS progression remains unclear. In the present study, ApoE-/- mice and endothelial cells line (HUVEC cells) were applied to investigate the effect of SIA on AS progression and its potential molecular mechanism. In vivo, mice were injected intraperitoneally with Neu5Ac (main form of SIA) to keep high-level SIA in circulation. ORO, H&E, and Masson staining were applied to detect the plaque progression. In vitro, HUVECs were treated with Neu5Ac at different times, CCK-8, RT-PCR, western blot, and immunoprecipitation methods were used to analyze its effects on endothelial function and the potential involved mechanism. Results from the present study showed that high plasma levels of Neu5Ac in ApoE-/- mice could aggravate the plaque areas as well as increase necrotic core areas and collagen fiber contents. Remarkably, Neu5Ac levels in circulation displayed a positive correlation with AS plaque areas. Furthermore, results from HUVECs showed that Neu5Ac inhibited cells viability in a time/dose-dependent manner, by then induced the activation of inflammation makers such as ICAM-1 and IL-1ß. Mechanism study showed that the activation of excessive autophagy medicated by SQSTM1/p62 displayed an important role in endothelium inflammatory injury. Neu5Ac could modify SQSTM1/p62 as a sialylation protein, and then increase its level with ubiquitin binding, further inducing ubiquitination degradation and being involved in the excessive autophagy pathway. Inhibition of sialylation by P-3Fax-Neu5Ac, a sialyltransferase inhibitor, reduced the binding of SQSTM1/p62 to ubiquitin. Together, these findings indicated that Neu5Ac increased SQSTM1/p62-ubiquitin binding through sialylation modification, thereby inducing excessive autophagy and subsequent endothelial injury. Inhibition of SQSTM1/p62 sialylation might be a potential strategy for preventing such disease with high levels of Neu5Ac in circulation.

MORG1 limits mTORC1 signaling by inhibiting Rag GTPases.

Autophagy, an important quality control and recycling process vital for cellular homeostasis, is tightly regulated. The mTORC1 signaling pathway regulates autophagy under conditions of nutrient availability and scarcity. However, how mTORC1 activity is fine-tuned during nutrient availability to allow basal autophagy is unclear. Here, we report that the WD-domain repeat protein MORG1 facilitates basal constitutive autophagy by inhibiting mTORC1 signaling through Rag GTPases. Mechanistically, MORG1 interacts with active Rag GTPase complex inhibiting the Rag GTPase-mediated recruitment of mTORC1 to the lysosome. MORG1 depletion in HeLa cells increases mTORC1 activity and decreases autophagy. The autophagy receptor p62/SQSTM1 binds to MORG1, but MORG1 is not an autophagy substrate. However, p62/SQSTM1 binding to MORG1 upon re-addition of amino acids following amino acid's depletion precludes MORG1 from inhibiting the Rag GTPases, allowing mTORC1 activation. MORG1 depletion increases cell proliferation and migration. Low expression of MORG1 correlates with poor survival in several important cancers.

Pyrogallol promotes growth arrest by activating the p53-mediated up-regulation of p21 and p62/SQSTM1-dependent degradation of ß-catenin in nonsmall cell lung cancer cells.

Pyrogallol (1,2,3-trihydroxybenzene), a polyphenolic natural compound, has attracted considerable attention with regard to its potential anticancer activity. However, further study is needed to elucidate the underlying mechanism related to the antiNSCLC activity of pyrogallol and provide a comprehensive theoretical basis for better clinical utilization of pyrogallol. Our current study aims to investigate the effects and potential underlying mechanisms of pyrogallol on the inhibition of NSCLC growth. Our results showed that pyrogallol treatment induced cell cycle arrest at the G2/M phase and apoptosis in two different NSCLC cell lines. Mechanistically, we found that the induction of cell cycle arrest in NSCLC cells at the G2/M phase by pyrogallol was due to the upregulation of p21 in a p53-dependent manner. And blockade of p53 and p21 effectively abolished the cell cycle arrest at the G2/M phase. Meanwhile, p53 inhibition has been found to abrogate the pyrogallol-induced apoptosis of the two NSCLC cells. Moreover, we revealed that the inhibitory effects of pyrogallol on ß-catenin signaling resulted from autophagy initiation depending on p53 activation, accompanied by an increase in p62/SQSTM1 expression, thus p62 subsequently interacting with ubiquitinated ß-catenin and facilitating autophagic destruction of ß-catenin. Furthermore, in vivo experiments demonstrated that pyrogallol exerted growth inhibition on NSCLC with low toxicity through the same molecular mechanism as observed in vitro. Our findings could contribute to the understanding of the mechanism by which pyrogallol negatively regulates NSCLC growth, which could be effective in treating NSCLC.