VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Circadian clock genes REV-ERBα regulates the secretion of IL-1ß in deciduous tooth pulp stem cells by regulating autophagy in the process of physiological root resorption of deciduous teeth.

Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth.

Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Neuronal activity promotes secretory autophagy for the extracellular release of α-synuclein.

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.

TAT-beclin1 treatment accelerates the development of atherosclerotic lesions in ApoE-deficient mice.

The importance of autophagy in atherosclerosis has garnered significant attention regarding the potential applications of autophagy inducers. However, the impact of TAT-Beclin1, a peptide inducer of autophagy, on the development of atherosclerotic plaques remains unclear. Single-cell omics analysis indicates a notable reduction in GAPR1 levels within fibroblasts, stromal cells, and macrophages during atherosclerosis. Tat-beclin1 (T-B), an autophagy-inducing peptide derived from Beclin1, could selectively bind to GAPR1, relieving its inhibition on Beclin1 and thereby augmenting autophagosome formation. To investigate its impact on atherosclerosic plaque progression, we established the ApoE-/- mouse model of carotid atherosclerotic plaques. Surprisingly, intravenous administration of Tat-beclin1 dramatically accelerated the development of carotid artery plaques. Immunofluorescence analysis suggested that macrophage aggregation and autophagosome formation within atherosclerotic plaques were significantly increased upon T-B treatment. However, immunofluorescence and transmission electron microscopy (TEM) analysis revealed a reduction in autophagy flux through lysosomes. In vitro, the interaction between T-B and GAPR1 was confirmed in RAW264.7 cells, resulting in the increased accumulation of p62/SQSTM1 and LC3-II in the presence of ox-LDL. Additionally, T-B treatment elevated the protein levels of p62/SQSTM1, LC3-II, and cleaved caspase 1, along with the secretion of IL-1ß in response to ox-LDL exposure. In summary, our study underscores that T-B treatment amplifies abnormal autophagy and inflammation, consequently exacerbating atherosclerotic plaque development in ApoE-/- mice.

TMEM9 activates Rab9-dependent alternative autophagy through interaction with Beclin1.

Transmembrane protein 9 (TMEM9) is a transmembrane protein that regulates lysosomal acidification by interacting with the v-type ATPase complex. However, the role of TMEM9 in the lysosome-dependent autophagy machinery has yet to be identified. In this study, we demonstrate that the lysosomal protein TMEM9, which is involved in vesicle acidification, regulates Rab9-dependent alternative autophagy through its interaction with Beclin1. The cytosolic domain of TMEM9 interacts with Beclin1 via its Bcl-2-binding domain. This interaction between TMEM9 and Beclin1 dissociates Bcl-2, an autophagy-inhibiting partner, from Beclin1, thereby activating LC3-independent and Rab9-dependent alternative autophagy. Late endosomal and lysosomal TMEM9 apparently colocalizes with Rab9 but not with LC3. Furthermore, we show that multiple glycosylation of TMEM9, essential for lysosomal localization, is essential for its interaction with Beclin1 and the activation of Rab9-dependent alternative autophagy. These findings reveal that TMEM9 recruits and activates the Beclin1 complex at the site of Rab9-dependent autophagosome to induce alternative autophagy.

The contribution and mechanism of hypoxia/USP19/Beclin-1 feed-forward loop in cervical cancer.

Cervical cancer ranks fourth in female mortality. Since the mechanisms for pathogenesis of cervical cancer are still poorly understood, the effective treatment options are lacking. Beclin-1 exhibits an inhibitory role in cervical cancer via suppressing the proliferation, invasion, and migration of cervical cancer cells. It is reported that USP19 removes the K11-linked ubiquitination of Beclin-1 to protect Beclin-1 from proteasomal degradation. Interestingly, we found that hypoxia induced a significant decrease of both Beclin-1 and USP19, suggesting that hypoxia could dually inhibit the protein level of Beclin-1 through a type 2 coherent feed-forward loop (C2-FFL, hypoxia ⊸ Beclin-1 integrating with hypoxia ⊸ USP19 → Beclin-1) to promote the occurrence and development of cervical cancer. Furthermore, mathematical modeling revealed that under the hypoxic environment of solid tumor, the hypoxia/USP19/Beclin-1 coherent feed-forward loop could significantly reduce the protein level of Beclin-1, greatly enhance the sensitivity of Beclin-1 to hypoxia, strikingly restrict the heterogeneity of Beclin-1, and contribute to the low positive rate of Beclin-1 in cervical cancer. It is expected to have significance for elucidating the underlying mechanisms of the occurrence and development of cervical cancer and to provide novel targets and strategies for prevention and treatment of cervical cancer.

Intracerebroventricular BDNF infusion may reduce cerebral ischemia/reperfusion injury by promoting autophagy and suppressing apoptosis.

Here, it was aimed to investigate the effects of intracerebroventricular (ICV) Brain Derived Neurotrophic Factor (BDNF) infusion for 7 days following cerebral ischemia (CI) on autophagy in neurons in the penumbra. Focal CI was created by the occlusion of the right middle cerebral artery. A total of 60 rats were used and divided into 4 groups as Control, Sham CI, CI and CI + BDNF. During the 7-day reperfusion period, aCSF (vehicle) was infused to Sham CI and CI groups, and BDNF infusion was administered to the CI + BDNF group via an osmotic minipump. By the end of the 7th day of reperfusion, Beclin-1, LC3, p62 and cleaved caspase-3 protein levels in the penumbra area were evaluated using Western blot and immunofluorescence. BDNF treatment for 7 days reduced the infarct area after CI, induced the autophagic proteins Beclin-1, LC3 and p62 and suppressed the apoptotic protein cleaved caspase-3. Furthermore, rotarod and adhesive removal test times of BDNF treatment started to improve from the 4th day, and the neurological deficit score from the 5th day. ICV BDNF treatment following CI reduced the infarct area by inducing autophagic proteins Beclin-1, LC3 and p62 and inhibiting the apoptotic caspase-3 protein while its beneficial effects were apparent in neurological tests from the 4th day.

Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

A study on the mechanism of Beclin-1 m6A modification mediated by catalpol in protection against neuronal injury and autophagy following cerebral ischemia.

OBJECTIVE: Catalpol (CAT) has various pharmacological activities and plays a protective role in cerebral ischemia. It has been reported that CAT played a protective role in cerebral ischemia by upregulaing NRF1 expression. Bioinformatics analysis reveals that NRF1 can be used as a transcription factor to bind to the histone acetyltransferase KAT2A. However, the role of KAT2A in cerebral ischemia remains to be studied. Therefore, we aimed to investigate the role of CAT in cerebral ischemia and its related mechanism. METHODS: In vitro, a cell model of oxygen and glucose deprivation/reperfusion (OGD/R) was constructed, followed by evaluation of neuronal injury and the expression of METTL3, Beclin-1, NRF1, and KAT2A. In vivo, a MCAO rat model was prepared by means of focal cerebral ischemia, followed by assessment of neurological deficit and brain injury in MCAO rats. Neuronal autophagy was evaluated by observation of autophagosomes in neurons or brain tissues by TEM and detection of the expression of LC3 and p62. RESULTS: In vivo, CAT reduced the neurological function deficit and infarct volume, inhibited neuronal apoptosis in the cerebral cortex, and significantly improved neuronal injury and excessive autophagy in MCAO rats. In vitro, CAT restored OGD/R-inhibited cell viability, inhibited cell apoptosis, LDH release, and neuronal autophagy. Mechanistically, CAT upregulated NRF1, NRF1 activated METTL3 via KAT2A transcription, and METTL3 inhibited Beclin-1 via m6A modification. CONCLUSION: CAT activated the NRF1/KAT2A/METTL3 axis and downregulated Beclin-1 expression, thus relieving neuronal injury and excessive autophagy after cerebral ischemia.

BECN1 mRNA expression in breast cancer tissue; significant correlation to tumor grade.

Breast cancer (BC) is a heterogenous disease with multiple pathways implicated in its development, progression, and drug resistance. Autophagy, a cellular process responsible for self-digestion of damaged organelles, had been recognized as eminent player in cancer progression and chemotherapeutic resistance. The haploinsufficiency of Beclin 1 (BECN1), autophagy protein, is believed to contribute to cancer pathogenesis and progression. In our study, we investigated the expression of BECN1 in a BC female Egyptian patient cohort, as well as its prognostic role through evaluating its association with disease free survival (DFS) after 2 years follow up and association of tumor clinicopathological features. Twenty frozen female BC tissue samples and 17 adjacent normal tissue were included and examined for the expression levels of BECN1. Although the tumor tissues showed lower expression 0.73 (0-8.95) than their corresponding normal tissues 1.02 (0.04-19.59), it was not statistically significant, p: 0.463. BECN1 expression was not associated with stage, nodal metastasis or tumor size, p:0.435, 0.541, 0.296, respectively. However, statistically significant negative correlation was found between grade and BECN1 mRNA expression in the studied cases, p:0.028. BECN1 expression had no statistically significant association with DFS, P = 0.944. However, we observed that triple negative (TNBC) cases had significantly lower DFS rate than luminal BC patients, p: 0.022, with mean DFS 19.0 months, while luminal BC patients had mean DFS of 23.41 months. Our study highlights the potential role of BECN1 in BC pathogenesis, showing that BECN1 expression correlates with poorer differentiation of BC, indicating its probable link with disease aggressiveness. DFS two years follow up showed that TNBC subtype remains associated with less favorable prognosis.

Beclin-1-Derived Peptide MP1 Attenuates Renal Fibrosis by Inhibiting the Wnt/ß-Catenin Pathway.

Renal fibrosis is distinguished by the abnormal deposition of extracellular matrix and progressive loss of nephron function, with a lack of effective treatment options in clinical practice. In this study, we discovered that the Beclin-1-derived peptide MP1 significantly inhibits the abnormal expression of fibrosis and epithelial-mesenchymal transition-related markers, including α-smooth muscle actin, fibronectin, collagen I, matrix metallopeptidase 2, Snail1, and vimentin both in vitro and in vivo. H&E staining was employed to evaluate renal function, while serum creatinine (Scr) and blood urea nitrogen (BUN) were used as main indices to assess pathologic changes in the obstructed kidney. The results demonstrated that daily treatment with MP1 during the 14-day experiment significantly alleviated renal dysfunction and changes in Scr and BUN in mice with unilateral ureteral obstruction. Mechanistic research revealed that MP1 was found to have a significant inhibitory effect on the expression of crucial components involved in both the Wnt/ß-catenin and transforming growth factor (TGF)-ß/Smad pathways, including ß-catenin, C-Myc, cyclin D1, TGF-ß1, and p-Smad/Smad. However, MP1 exhibited no significant impact on either the LC3II/LC3I ratio or P62 levels. These findings indicate that MP1 improves renal physiologic function and mitigates the fibrosis progression by inhibiting the Wnt/ß-catenin pathway. Our study suggests that MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis. SIGNIFICANCE STATEMENT: This study indicated that the Beclin-1-derived peptide MP1 effectively mitigated renal fibrosis induced by unilateral ureteral obstruction through inhibiting the Wnt/ß-catenin pathway and transforming growth factor-ß/Smad pathway, thereby improving renal physiological function. Importantly, unlike other Beclin-1-derived peptides, MP1 exhibited no significant impact on autophagy in normal cells. MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis focusing on Beclin-1 derivatives and Wnt/ß-catenin pathway.

SIAH1 modulates antiviral immune responses by targeting deubiquitinase USP19.

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.

Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Enhancing endometrial receptivity: the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells.

Inadequate endometrial receptivity often results in embryo implantation failure and miscarriage. Human chorionic gonadotropin (hCG) is a key signaling molecule secreted during early embryonic development, which regulates embryonic maternal interface signaling and promotes embryo implantation. This study aimed to examine the impact of hCG on endometrial receptivity and its underlying mechanisms. An exploratory study was designed, and endometrial samples were obtained from women diagnosed with simple tubal infertility or male factor infertile (n = 12) and recurrent implantation failure (RIF, n = 10). Using reverse transcription-quantitative PCR and western blotting, luteinizing hormone (LH)/hCG receptor (LHCGR) levels and autophagy were detected in the endometrial tissues. Subsequently, primary endometrial stromal cells (ESCs) were isolated from these control groups and treated with hCG to examine the presence of LHCGR and markers of endometrial receptivity (HOXA10, ITGB3, FOXO1, LIF, and L-selectin ligand) and autophagy-related factors (Beclin1, LC3, and P62). The findings revealed that the expressions of receptivity factors, LHCGR, and LC3 were reduced in the endometrial tissues of women with RIF compared with the control group, whereas the expression of P62 was elevated. The administration of hCG to ESCs specifically activated LHCGR, stimulating an increase in the endometrial production of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligands. Furthermore, when ESCs were exposed to 0.1 IU/mL hCG for 72 h, the autophagy factors Beclin1 and LC3 increased within the cells and P62 decreased. Moreover, the apoptotic factor Bax increased and Bcl-2 declined. However, when small interfering RNA was used to knock down LHCGR, hCG was less capable of controlling endometrial receptivity and autophagy molecules in ESCs. In addition, hCG stimulation enhanced the phosphorylation of ERK1/2 and mTOR proteins. These results suggest that women with RIF exhibit lower levels of LHCGR and compromised autophagy function in their endometrial tissues. Thus, hCG/LHCGR could potentially improve endometrial receptivity by modulating autophagy and apoptosis.

High expression of TBRG4 in relation to unfavorable outcome and cell ferroptosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of malignant liver tumor with poor prognosis. In this study, we investigated the expression of transforming growth factor beta regulator 4 (TBRG4) in HCC and its effects on the proliferation, invasion, and metastasis of HCC cells, and analyzed the possible molecular mechanisms. METHOD: Downloading the expression and clinical information of HCC samples in the TCGA database, analyzing the expression differences of TBRG4 by bioinformatics methods, analyzing the clinical relevance and prognostic significance. Performing GO, KEGG and GSEA enrichment analysis on the TBRG4-related gene set in patient HCC tissues. Applying cell counting, scratch test and Transwell experiment to study the biological function of TBRG4 in HCC. Mitochondrial membrane potential, apoptosis and ROS levels were evaluated to assess cell iron death. Western blot, RT-PCR, laser confocal microscopy and co-immunoprecipitation were used to detect and analyze the downstream signaling pathways and interacting molecules of TBRG4. RESULTS: Bioinformatics analysis revealed that TBRG4 was abnormally highly expressed in HCC tumor tissues and was associated with poor prognosis and metastasis in HCC patients. GO and KEGG functional enrichment analysis showed that TBRG4 was related to oxidative stress and NADH dehydrogenase (ubiquinone) activity. GSEA enrichment analysis showed that TBRG4 was associated with Beta catenin independent wnt signaling and B cell receptor. Functional experiments confirmed that knocking down TBRG4 could inhibit the proliferation, migration, and invasion of HCC cells. Mechanistically, TBRG4 inhibited the function of HCC cells through the DDX56/p-AKT/GSK3ß signaling pathway. In addition, interference with TBRG4 expression could reduce the mitochondrial membrane potential and accumulate ROS in HCC cells, leading to increased ferroptosis. Co-IP analysis showed that TBRG4 specifically bound to Beclin1. CONCLUSION: TBRG4 is highly expressed in HCC tumor tissues and is associated with poor prognosis. It may regulate the proliferation, invasion, and metastasis of HCC cells through the DDX56/p-AKT/GSK3ß signaling pathway. TBRG4 may interact with Beclin1 to regulate the ferroptosis of HCC cells.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

GSK-3ß as a Potential Coordinator of Anabolic and Catabolic Pathways in Hepatitis C Virus Insulin Resistance.

INTRODUCTION: Chronic hepatitis C infection can result in insulin resistance (IR). We have previously shown that it occurs through the interaction of pathways for glucose homeostasis, insulin signaling, and autophagy. But it is not known how soon the pathways are activated and how IR is related to the signals generated by catabolic and anabolic conditions occurring in infected cells. We have extended our studies to a cell culture system mimicking acute infection and to downstream pathways involving energy-sensor AMPK and nutrient-sensor mTOR that are active in catabolic and anabolic processes within the infected cells. METHODS: Huh7 liver cells in culture were infected with hepatitis C virus (HCV). We performed proteomics analysis of key proteins in infected cells by Western blotting and IP experiments, with or without IFNα exposure as a component of conventional therapeutic strategy. RESULTS: We present evidence that (a) IRS-1 Ser312, Beclin-1, protein conjugate Atg12-Atg5 or GS Ser641 are up-regulated early in infection presumably by activating the same pathways as utilized for persistent infection; (b) Bcl-XL, an inhibitor of both autophagy and apoptosis, is present in a core complex with IRS-1 Ser312 and Beclin-1 during progression of IR; (c) AMPK level remains about the same in infected cells where it is activated by phosphorylation at Thr172 concomitant with increased autophagy, a hallmark of catabolic conditions; (d) an mTOR level that promotes anabolism is increased rather than decreased under an expanded autophagy; (e) hypophosphorylation of translational repressor 4E-BP1 downstream of mTOR is suggestive of reduced protein synthesis; and (f) ß-catenin, is up-regulated but not phosphorylated suggesting indirectly our previous contention that its kinase, GSK-3ß, is mostly in an inactive state. CONCLUSION: We report that in the development of IR following chronic infection, anabolic and catabolic pathways are activated early, and the metabolic interaction occurs possibly in a core complex with IRS-1 Ser312, Beclin-1, and autophagy inhibitor Bcl-XL. Induction of autophagy is usually controlled by a two-edged mechanism acting in opposition under anabolic and catabolic conditions by AMPK/mTOR/4E-BP1 pathway with GSK-3ß-mediated feedback loops. However, we have observed an up-regulation of mTOR along with an up-regulation of AMPK caused by HCV infection is a deviation from the normal scenario described above which might be of therapeutic interest.

Functional identification of two HMGB1 paralogues provides insights into autophagic machinery in teleost.

High mobility group box 1 (HMGB1) is a multifunctional regulator that plays different roles in various physiological and pathological processes including cell development, autophagy, inflammation, tumor metastasis, and cell death based on its cellular localization. Unlike mammalian HMGB1, two HMGB1 paralogues (HMGB1a and HMGB1b) have been found in fathead minnow and other fish species and its function as an inflammatory cytokine has been well investigated. However, the role of fish HMGB1 in autophagy regulation has not been well clarified. In the present study, we generated HMGB1 paralogues single (HMGB1a-/- and HMGB1b-/-) and double knockout (DKO) epithelioma papulosum cyprini (EPC) cells from fathead minnow by CRISPR/Cas9 system, and the knockout efficiency of these genes was verified at both gene and protein levels. In this context, the effects of HMGB1 gene knockout on the protein expression of microtubule-associated protein 1 light chain 3 II (LC3-II), an autophagy marker, were determined, showing that single knockout of two HMGB1 paralogues significantly decreased the expression of LC3-II, and these inhibitory effects were further amplified in HMGB1 DKO cells under both basal and rapamycin treatment conditions, indicating the role of two HMGB1 paralogues in fish autophagy. In agreement with this notion, overexpression of HMGB1a or HMGB1b with Flag-tag markedly upregulated LC3-II protein expression. Interestingly, overexpressing two paralogues distributed in both cytoplasm and nucleus. Finally, the role of HMGB1-mediated autophagy was further explored, finding that HMGB1 could interact with Beclin1, a key initiation factor of autophagy. Taken together, these findings highlighted the role of HMGB1 paralogues as the autophagy regulator and increased our understanding of autophagic machinery in teleost.

Anesthesia/surgery-induced learning and memory dysfunction by inhibiting mitophagy-mediated NLRP3 inflammasome inactivation in aged mice.

Postoperative cognitive dysfunction (POCD) is a common postoperative complication, not only affects the quality of life of the elderly and increases the mortality rate, but also brings a greater burden to the family and society. Previous studies demonstrated that Nod-like receptor protein 3 (NLRP3) inflammasome participates in various inflammatory and neurodegenerative diseases. However, possible mitophagy mechanism in anesthesia/surgery-elicited NLRP3 inflammasome activation remains to be elucidated. Hence, this study clarified whether mitophagy dysfunction is related to anesthesia/surgery-elicited NLRP3 inflammasome activation. POCD model was established in aged C57BL/6 J mice by tibial fracture fixation under isoflurane anesthesia. Morris Water Maze (MWM) was used to evaluate learning and memory abilities. We found that in vitro experiments, lipopolysaccharide (LPS) significantly facilitated NLRP3 inflammasome activation and mitophagy inhibition in BV2 cells. Rapamycin restored mitophagy and improved mitochondrial function, and inhibited NLRP3 inflammasome activation induced by LPS. In vivo experiments, anesthesia and surgery caused upregulation of hippocampal NLRP3, caspase recruitment domain (ASC) and interleukin-1ß (IL-1 ß), and downregulation of microtubule-associated protein light chain 3II (LC3II) and Beclin1 in aged mice. Olaparib inhibited anesthesia/surgery-induced NLRP3, ASC, and IL-1ß over-expression in the hippocampus, while upregulated the expression of LC3II and Beclin1. Furthermore, Olaparib improved cognitive impairment in older mice. These results revealed that mitophagy was involved in NLRP3 inflammasome-mediated anesthesia/surgery-induced cognitive deficits in aged mice. Overall, our results suggested that mitophagy was related in NLRP3 inflammasome-induced cognitive deficits after anesthesia and surgery in aged mice. Activating mitophagy may have clinical benefits in the prevention of cognitive impairment induced by anesthesia and surgery in elderly patients.