Xiao-Er-Kang-Du capsules regulate autophagy against the influenza B virus (Victoria strain) through the mTOR/ULK1/Beclin1/VPS34 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiao-er-kang-du (XEKD) capsule is a Chinese herbal formula used for treatment of upper respiratory tract infection caused by various viruses in pediatric patients in China. XEKD is used clinically for the treatment of influenza-like symptoms, including fever, chills, cough, stuffy and runny nose, headache, and sore throat, with remarkable efficacy. However, the pharmacologic mechanism of XEKD against influenza B virus (IBV) infection is unclear. AIM OF THE STUDY: The main purpose of the present work is to explore the curative effect as well as possible mechanisms of XEKD against influenza B virus (IBV) (Victoria strain). MATERIALS AND METHODS: Both in vivo and in vitro experiments were performed to confirm the antiviral properties of XEKD. High-performance liquid chromatography was used to analyze the active components and assess the stability of XEKD. In addition, the mechanism of action of XEKD against IBV (Victoria) was investigated by western blot, immunofluorescence, and immunohistochemical analyses, in addition to confocal fluorescence microscopy. RESULTS: The findings revealed that XEKD demonstrated antiviral effects against IBV infection in both in vivo and in vitro via the mTOR/ULK1/Beclin1/VPS34 pathway and promote cellular autophagy to mitigate IBV-induced lung tissue damage. The results of this work are expected to lead to a deeper understanding of the mechanism underlying the effect of the XEKD capsule against IBV infections. CONCLUSIONS: IBV infection was found to inhibit autophagy, which exacerbated inflammatory damage. XEKD regulates autophagy through the mTOR/ULK1/Beclin1/VPS34 pathway and exerts antiviral effects, thereby laying a foundation for further development of XEKD as a potential therapeutic against IBV infection.

Regulatory Mechanisms Governing the Autophagy-Initiating VPS34 Complex and Its inhibitors.

VPS34 is a crucial protein in cells, essential for handling cellular stress through its involvement in autophagy and endocytosis. This protein functions as a Class III phosphatidylinositol 3-kinase, producing phosphatidylinositol 3-phosphate, which is necessary for autophagy and vesicle trafficking. Additionally, VPS34 forms two mutually exclusive complexes, each playing a vital role in autophagy and endocytic sorting. These complexes share common subunits, including VPS15, VPS34, and Beclin 1, with complex I having ATG14 as a specific subunit. Due to its association with various human diseases, regulation of the VPS34 complex I has garnered significant interest, emerging as a potential therapeutic target for drug discovery. Summaries of the structure, function of VPS34 complexes, and developed VPS34 inhibitors have been provided, along with discussions on the regulation mechanism of VPS34, particularly in relation to the initiation complex I of autophagy. This offers valuable insights for treating autophagy-related diseases.

Abnormal expression of autophagy proteins in the duodenum of patients with functional dyspepsia: A preliminary study.

BACKGROUND AND STUDY AIMS: Functional dyspepsia (FD) is a common disease with an unclear pathology. Autophagy is associated with inflammation and has been proposed to play a role in the development of FD. This study aimed to evaluate expression of the autophagy proteins beclin1 and p62/SQSTM1 in patients with FD. PATIENTS AND METHODS: Duodenal mucosal tissues were collected from 10 patients with FD and 10 asymptomatic controls. The extent of autophagy was determined by examining expression levels of beclin1 and p62/SQSTM1 using quantitative polymerase chain reaction and immunohistochemistry techniques. RESULTS: Lower expression levels of beclin1 protein were detected in the duodenal bulb (D1) and the second portion of the duodenum (D2) in patients with FD compared with asymptomatic controls. Higher levels of p62 protein were expressed in D1 in patients with FD than in controls. No differences in mRNA expression of beclin1 and p62 were observed between patients with FD and controls. CONCLUSION: Abnormal autophagy was involved in FD, which may be associated with the pathogenesis of FD.

Investigation of Urotensin II expression in placenta and umbilical cord in pregnancies with intrauterine growth restriction by histological and biochemical methods.

OBJECTIVE: In this study, it was aimed to investigate Urotensin II in intrauterine growth restriction (IUGR) and its connection to autophagy and/or apoptosis in placenta and umbilical cord by immunohistochemical and biochemical methods. MATERIALS AND METHODS: The study included 30 healthy pregnant women and 30 pregnant women with IUGR, aged 19-45, at Atatürk University Gynecology Clinic. Samples were collected from placenta, umbilical cord, maternal blood, and umbilical cord blood during delivery. Histopathological examination was carried out on placenta and umbilical cord, and UTII, Beclin 1, and caspase 3 expressions were analyzed in these tissues. Biochemical analysis was performed on maternal and umbilical cord serum samples. RESULTS: In healthy placentas, normal villus formation was seen, but those with IUGR showed accelerated villus maturation, causing inadequate nutrition and development. IUGR placentas had fibrin deposition, villous edema, syncytial nodes increase, and intervillous distance. Umbilical cords of IUGR group had differences in vessel wall thickness, arterial lumens, and vessel number. Higher levels of UTII, Beclin 1, and caspase 3 were found in IUGR placenta and cord. Beclin 1 and caspase 3 levels were significantly higher in IUGR group compared to controls, while UTII levels were not significantly different in maternal and cord serums. CONCLUSION: As a result of our findings, UTII increase in placenta and umbilical cord may lead to IUGR formation by inducing autophagy and apoptosis.

Downregulating miR-432-5p exacerbates adriamycin-induced cardiotoxicity via activating the RTN3 signaling pathway.

BACKGROUND: Adriamycin (ADR) is a widely used chemotherapy drug in clinical practice and it causes toxicity in the myocardium affecting its clinical use. miR-432-5p is a miRNA primarily expressed in myocardial cells and has a protective effect in the myocardium. We aim to explore the protective effect of miR-432-5p on ADR-caused impaired mitochondrial ATP metabolism and endoplasmic reticulum stress (ERs). METHOD: The primary cardiomyocytes were obtained from neonatal mice and the ADR was added to cells, meanwhile, a mice model was constructed through intravenous ADR challenge, and expression levels of miR-432-5p were examined. Subsequently, the miR-432-5p was introduced in vitro and in vivo to explore its effect on the activity of mitochondrial ATP synthesis, autophagy, and ER stress. The bioinformatics analysis was performed to explore the target of miR-432-5p. RESULTS: ADR decreased the expression of miR-432-5p in cardiomyocytes. It also decreases mitochondrial ATP production and activates the ER stress pathway by increasing the expression of LC3B, Beclin 1, cleaved caspase 3, and induces cardiac toxicity. miR-432-5p exogenous supplementation can reduce the cardiotoxicity caused by ADR, and its protective effect on cardiomyocytes depends on the down-regulation of the RTN3 signaling pathway in ER. CONCLUSION: ADR can induce the low expression of miR-432-5p, and activate the RTN3 pathway in ER, increase the expression of LC3B, Beclin 1, cleaved caspase 3, CHOP, and RTN3, and induce cardiac toxicity.

Suppression of autophagy induces senescence in the heart.

Aging is a critical risk factor for heart disease, including ischemic heart disease and heart failure. Cellular senescence, characterized by DNA damage, resistance to apoptosis and the senescence-associated secretory phenotype (SASP), occurs in many cell types, including cardiomyocytes. Senescence precipitates the aging process in surrounding cells and the organ through paracrine mechanisms. Generalized autophagy, which degrades cytosolic materials in a non-selective manner, is decreased during aging in the heart. This decrease causes deterioration of cellular quality control mechanisms, facilitates aging and negatively affects lifespan in animals, including mice. Although suppression of generalized autophagy could promote senescence, it remains unclear whether the suppression of autophagy directly stimulates senescence in cardiomyocytes, which, in turn, promotes myocardial dysfunction in the heart. We addressed this question using mouse models with a loss of autophagy function. Suppression of general autophagy in cardiac-specific Atg7 knockout (Atg7cKO) mice caused accumulation of senescent cardiomyocytes. Induction of senescence via downregulation of Atg7 was also observed in chimeric Atg7 cardiac-specific KO mice and cultured cardiomyocytes in vitro, suggesting that the effect of autophagy suppression upon induction of senescence is cell autonomous. ABT-263, a senolytic agent, reduced the number of senescent myocytes and improved cardiac function in Atg7cKO mice. Suppression of autophagy and induction of senescence were also observed in doxorubicin-treated hearts, where reactivation of autophagy alleviated senescence in cardiomyocytes and cardiac dysfunction. These results suggest that suppression of general autophagy directly induces senescence in cardiomyocytes, which in turn promotes cardiac dysfunction.

Trypanosoma cruzi Vps34 colocalizes with Beclin1 and plays a role in parasite invasion of the host cell by modulating the expression of a sub-group of trans-sialidases.

Trypanosoma cruzi, the etiological agent of Chagas' disease, can infect both phagocytic and non-phagocytic cells. T. cruzi gp82 and gp90 are cell surface proteins belonging to Group II trans-sialidases known to be involved in host cell binding and invasion. Phosphatidylinositol kinases (PIK) are lipid kinases that phosphorylate phospholipids in their substrates or in themselves, regulating important cellular functions such as metabolism, cell cycle and survival. Vps34, a class III PIK, regulates autophagy, trimeric G-protein signaling, and the mTOR (mammalian Target of Rapamycin) nutrient-sensing pathway. The mammalian autophagy gene Beclin1 interacts to Vps34 forming Beclin 1-Vps34 complexes involved in autophagy and protein sorting. In T. cruzi epimastigotes, (a non-infective replicative form), TcVps34 has been related to morphological and functional changes associated to vesicular trafficking, osmoregulation and receptor-mediated endocytosis. We aimed to characterize the role of TcVps34 during invasion of HeLa cells by metacyclic (MT) forms. MTs overexpressing TcVps34 showed lower invasion rates compared to controls, whilst exhibiting a significant decrease in gp82 expression in the parasite surface. In addition, we showed that T. cruzi Beclin (TcBeclin1) colocalizes with TcVps34 in epimastigotes, thus suggesting the formation of complexes that may play conserved cellular roles already described for other eukaryotes.

Spexin inhibits excessive autophagy-induced ferroptosis to alleviate doxorubicin-induced cardiotoxicity by upregulating Beclin 1.

BACKGROUND AND PURPOSE: Doxorubicin is widely used in the treatment of malignant tumours, but doxorubicin-induced cardiotoxicity severely limits its clinical application. Spexin is a neuropeptide that acts as a novel biomarker in cardiovascular disease. However, the effects of spexin on doxorubicin-induced cardiotoxicity is unclear. EXPERIMENTAL APPROACH: We established a model of doxorubicin-induced cardiotoxicity both in vivo and in vitro. Levels of cardiac damage in mice was assessed through cardiac function assessment, determination of serum cardiac troponin T and CKMB levels and histological examination. CCK8 and PI staining were used to assess the doxorubicin-induced toxicity in cultures of cardiomyocytes in vitro. Ferroptosis was assessed using FerroOrange staining, determination of MDA and 4-HNE content and ferroptosis-associated proteins SLC7A11 and GPX4. Mitochondrial membrane potential and lipid peroxidation levels were measured using TMRE and C11-BODIPY 581/591 probes, respectively. Myocardial autophagy was assessed by expression of P62 and Beclin1. KEY RESULTS: Spexin treatment improved heart function of mice with doxorubicin-induced cardiotoxicity, and attenuated doxorubicin-induced cardiotoxicity by decreasing iron accumulation, abnormal lipid metabolism and inhibiting ferroptosis. Interestingly, doxorubicin caused excessive autophagy in cardiomyocyte in culture, which could be alleviated by treatment with spexin. Knockdown of Beclin 1 eliminated the protective effects of spexin in mice with DIC. CONCLUSION AND IMPLICATIONS: Spexin ameliorated doxorubicin-induced cardiotoxicity by inhibiting excessive autophagy-induced ferroptosis, suggesting that spexin could be a drug candidate against doxorubicin-induced cardiotoxicity. Beclin 1 might be critical in mediating the protective effect of spexin against doxorubicin-induced cardiotoxicity.

Human ß-defensin-1 activates autophagy in human colon cancer cells via regulation of long non-coding RNA TCONS_00014506.

Macroautophagy (hereafter referred to as autophagy) is a prosurvival mechanism for the clearance of damaged cellular components, specifically related to exposure to various stressors such as starvation, excessive ethanol intake, and chemotherapy. This editorial reviews and comments on an article by Zhao et al, to be published in World J Gastrointestinal Oncology in 2024. Based on various molecular biology methodologies, they found that human ß-defensin-1 reduced the proliferation of colon cancer cells, which was associated with the inhibition of the mammalian target of rapamycin, resulting in autophagy activation. The activation of autophagy is evidenced by increased levels of Beclin1 and LC3II/I proteins and mediated by the upregulation of long non-coding RNA TCONS_00014506. Our study discusses the impact of autophagy activation and mechanisms of autophagy, including autophagic flux, on cancer cells. Additionally, we emphasize the importance of describing the detailed methods for isolating long noncoding RNAs TCONS_00014506. Our review will benefit the scientific community and improve the overall clarity of the paper.

Insulin-like growth factor-1 reduces cardiac autosis through decreasing AMPK/FOXO1 signaling and Na+/K+-ATPase-Beclin-1 interaction.

Introduction: Insulin-like growth factor-1 (IGF-1) promotes survival and inhibits cardiac autophagy disruption. Methods: Male Wistar rats were treated with IGF-1 (50 µg/kg), and 24 h after injection hearts were excised. The level of interaction between Beclin-1 and the α1 subunit of sodium/potassium-adenosine triphosphates (Na+/K+-ATPase), and phosphorylated forms of IGF-1 receptor/insulin receptor (IGF-1R/IR), forkhead box protein O1 (FOXO1) and AMP-activated protein kinase (AMPK) were measured. Results: The results indicate that IGF-1 decreased Beclin-1's association with Na+/K+-ATPase (p < 0.05), increased IGF-1R/IR and FOXO1 phosphorylation (p < 0.05), and decreased AMPK phosphorylation (p < 0.01) in rats' hearts. Conclusions: The new IGF-1 therapy may control autosis and minimize cardiomyocyte mortality.

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.

Immunohistochemistry analysis of autophagy-related proteins Beclin-1, p62/SQSTM1, and LC3B in breast carcinoma progression to bone metastasis.

Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.

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.

Catharanthine, an anticancer vinca alkaloid: an in silico and in vitro analysis of the autophagic system as the major mechanism of cell death in liver HepG2 cells.

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (-10.7 KJ/mol-1) than catharanthine (-7.3 KJ/mol-1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR.

Ranolazine ameliorates T1DM-induced testicular dysfunction in rats; role of NF-κB/TXNIP/GSDMD-N/IL-18/Beclin-1 signaling pathway.

Approximately 90% of diabetic males have varying degrees of testicular dysfunction. The current study investigates the possible beneficial consequences of ranolazine against T1DM-induced testicular dysfunction in rats. Thirty-two male Sprague Dawley rats were assorted into 4 groups; normal, diabetic (single 50 mg/kg STZ, I.P.) and ranolazine (40 and 80 mg/kg, orally). The present investigation revealed that the hypoglycemic impact of ranolazine significantly improved the testicular weight and body weight of the final rats, as well as the concentration of blood testosterone, sperm count, and viability, all of which were associated with STZ-induced testicular dysfunction. Furthermore, as demonstrated by elevated reduced glutathione (GSH) activity and lowered malondialdehyde (MDA) levels, diabetic rats administered ranolazine showed a noteworthy improvement in the oxidant/antioxidant ratio. Furthermore, a substantial rise in beclin-1 concentration was seen in conjunction with a significant decrease in thioredoxin-interacting protein (TXNIP) and interleukin-18 (IL-18) concentrations when ranolazine was administered. Although ranolazine exhibited a reduction in inflammation as seen by lower expression of nuclear factor-κB (NF-κB) and cluster of differentiation (CD68) in the testicles, these biochemical findings were validated by improvements in the morphological and histopathological outcomes of both the pancreatic and testicular tissues. In conclusion, daily oral administration of ranolazine (40 and 80 mg/kg) for 8 weeks could be a promising therapy for T1DM-induced testicular dysfunction through its dose-dependent anti-oxidant and anti-inflammatory effects.

YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages.

BACKGROUND: YTHDC1, a key m(6)A nuclear reader, plays a crucial role in regulating mRNA splicing, export, and stability. However, the functional significance and regulatory mechanisms of YTHDC1 in inflammatory bowel disease (IBD) remain to be explored. METHODS: We established a dextran sulfate sodium (DSS)-induced murine colitis model in vivo and LPS/IFN-γ-stimulated macrophage inflammation in vitro. The expression of YTHDC1 was determined. Colocalization of YTHDC1 and macrophages was assayed by immunofluorescence staining. LV-YTHDC1 or shYTHDC1 lentiviruses were applied for YTHDC1 overexpression or inhibition. For NF-κB inhibition, JSH-23 was utilized. The interaction of YTHDC1 and Beclin1 mRNA was determined by RIP, and the m6A modification of Beclin1 was confirmed by MeRIP. RESULTS: In DSS-induced colitis and LPS/IFN-γ-treated RAW264.7 macrophages, we observed a significant downregulation of YTHDC1. Overexpression of YTHDC1 resulted in decreased levels of iNOS, CD86, and IL-6 mRNA, along with inhibited NF-κB activation in LPS/IFN-γ-treated RAW264.7 cells. Conversely, downregulation of YTHDC1 promoted iNOS expression and inhibited autophagy. Additionally, the effect of YTHDC1 knockdown on CD86 and IL-6 mRNA induced by LPS/IFN-γ was abolished by the NF-κB inhibitor JSH-23. Mechanistically, YTHDC1 interacted with Beclin1 mRNA, thereby stabilizing Beclin1 mRNA and enhancing Beclin1 expression and autophagy. These effects ultimately led to the inhibition of NF-κB signaling in LPS/IFN-γ-challenged macrophages. CONCLUSIONS: YTHDC1 inhibited the macrophage-mediated inflammatory response by stabilizing Beclin1 mRNA, which may be a potential therapeutic target for the treatment of IBD.

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.

PKD2 regulates autophagy and forms a protein complex with BECN1 at the primary cilium of hypothalamic neuronal cells.

The primary cilium, hereafter cilium, is an antenna-like organelle that modulates intracellular responses, including autophagy, a lysosomal degradation process essential for cell homeostasis. Dysfunction of the cilium is associated with impairment of autophagy and diseases known as "ciliopathies". The discovery of autophagy-related proteins at the base of the cilium suggests its potential role in coordinating autophagy initiation in response to physiopathological stimuli. One of these proteins, beclin-1 (BECN1), it which is necessary for autophagosome biogenesis. Additionally, polycystin-2 (PKD2), a calcium channel enriched at the cilium, is required and sufficient to induce autophagy in renal and cancer cells. We previously demonstrated that PKD2 and BECN1 form a protein complex at the endoplasmic reticulum in non-ciliated cells, where it initiates autophagy, but whether this protein complex is present at the cilium remains unknown. Anorexigenic pro-opiomelanocortin (POMC) neurons are ciliated cells that require autophagy to maintain intracellular homeostasis. POMC neurons are sensitive to metabolic changes, modulating signaling pathways crucial for controlling food intake. Exposure to the saturated fatty acid palmitic acid (PA) reduces ciliogenesis and inhibits autophagy in these cells. Here, we show that PKD2 and BECN1 form a protein complex in N43/5 cells, an in vitro model of POMC neurons, and that both PKD2 and BECN1 locate at the cilium. In addition, our data show that the cilium is required for PKD2-BECN1 protein complex formation and that PA disrupts the PKD2-BECN1 complex, suppressing autophagy. Our findings provide new insights into the mechanisms by which the cilium controls autophagy in hypothalamic neuronal cells.

VCP/p97 UFMylation stabilizes BECN1 and facilitates the initiation of autophagy.

Macroautophagy/autophagy is essential for the degradation and recycling of cytoplasmic materials. The initiation of this process is determined by phosphatidylinositol-3-kinase (PtdIns3K) complex, which is regulated by factor BECN1 (beclin 1). UFMylation is a novel ubiquitin-like modification that has been demonstrated to modulate several cellular activities. However, the role of UFMylation in regulating autophagy has not been fully elucidated. Here, we found that VCP/p97 is UFMylated on K109 by the E3 UFL1 (UFM1 specific ligase 1) and this modification promotes BECN1 stabilization and assembly of the PtdIns3K complex, suggesting a role for VCP/p97 UFMylation in autophagy initiation. Mechanistically, VCP/p97 UFMylation stabilizes BECN1 through ATXN3 (ataxin 3)-mediated deubiquitination. As a key component of the PtdIns3K complex, stabilized BECN1 facilitates assembly of this complex. Re-expression of VCP/p97, but not the UFMylation-defective mutant, rescued the VCP/p97 depletion-induced increase in MAP1LC3B/LC3B protein expression. We also showed that several pathogenic VCP/p97 mutations identified in a variety of neurological disorders and cancers were associated with reduced UFMylation, thus implicating VCP/p97 UFMylation as a potential therapeutic target for these diseases. Abbreviation: ATG14:autophagy related 14; Baf A1:bafilomycin A1;CMT2Y: Charcot-Marie-Toothdisease, axonal, 2Y; CYB5R3: cytochromeb5 reductase 3; DDRGK1: DDRGK domain containing 1; DMEM:Dulbecco'smodified Eagle's medium;ER:endoplasmic reticulum; FBS:fetalbovine serum;FTDALS6:frontotemporaldementia and/or amyotrophic lateral sclerosis 6; IBMPFD1:inclusion bodymyopathy with early-onset Paget disease with or withoutfrontotemporal dementia 1; LC-MS/MS:liquid chromatography tandem mass spectrometry; MAP1LC3B/LC3B:microtubule associated protein 1 light chain 3 beta; MS: massspectrometry; NPLOC4: NPL4 homolog, ubiquitin recognition factor;PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3;PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K:phosphatidylinositol 3-kinase; RPL26: ribosomal protein L26; RPN1:ribophorin I; SQSTM1/p62: sequestosome 1; UBA5: ubiquitin likemodifier activating enzyme 5; UFC1: ubiquitin-fold modifierconjugating enzyme 1; UFD1: ubiquitin recognition factor in ERassociated degradation 1; UFL1: UFM1 specific ligase 1; UFM1:ubiquitin fold modifier 1; UFSP2: UFM1 specific peptidase 2; UVRAG:UV radiation resistance associated; VCP/p97: valosin containingprotein; WT: wild-type.