Promotes Autophagosome Formation Research Articles

Phillyrin promotes autophagosome formation in A53T-αSyn-induced Parkinson's disease model via modulation of REEP1

BackgroundThe preservation of autophagosome formation presents a promising strategy for tackling neurological disorders, such as Parkinson's disease (PD). Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) serve not only as a focal point linked to various neurological disorders but also play a crucial role in supporting the biogenesis of autophagosomes. PurposeThis investigation aimed to elucidate the neuroprotective properties of phillyrin against PD and its underlying mechanisms in promoting autophagosome formation. MethodsER and mitochondria co-localization was assessed via fluorescent staining. Annexin V-fluorescein isothiocyanate (FITC) fluorescence was employed to quantify accessible cardiolipin (CL) on mitochondrial surfaces. The levels of CL within the MAM fraction of SH-SY5Y cells were evaluated using a CL probe assay kit. Monodansylcadaverine staining was utilized to detect autophagosome formation in SH-SY5Y cells. In an A53T-alpha-synuclein (αSyn)-induced PD mouse model, the anti-PD properties of phillyrin were assessed using open field, pole climbing, and rotarod tests, as well as immunohistochemistry staining of TH+ neurons in the brain sections. ResultsIn A53T-αSyn-treated SH-SY5Y cells, phillyrin facilitated autophagosome formation by suppressing CL externalization and restoring MAM integrity. Phillyrin enhanced the localization of receptor expression-enhancing protein 1 (REEP1) within MAM and mitochondria, bolstering MAM formation. Increased REEP1 levels in mitochondria, attributed to phillyrin, enhanced the interaction between REEP1 and NDPK-D, thereby reducing CL externalization. Furthermore, phillyrin exhibited a dose-dependent enhancement of motor function in mice, accompanied by an increase in the abundance of dopaminergic neurons within the substantia nigra. ConclusionsThese findings illuminate phillyrin's ability to enhance MAM formation through upregulation of REEP1 expression within MAM, while concurrently attenuating CL externalization via the REEP1-NDPK-D interaction. These mechanisms bolster autophagosome biogenesis, offering resilience against A53T-αSyn-induced PD. Thus, our study advances the understanding of phillyrin's complex mechanisms and underscores its potential as a therapeutic approach for PD, opening new avenues in natural product pharmacology.

NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

Autophagy is closely related to the occurrence and development of human malignancies; however, the detailed mechanisms underlying autophagy in cervical cancer require further investigation. Previously, we found that the ectopic expression of NCAPH, a regulatory subunit of condensed protein complexes, significantly enhanced the proliferation of tumor cells; however, the underlying mechanisms were unclear. Here, we revealed that NCAPH is a novel autophagy-associated protein in cervical cancer that promotes cell proliferation by inhibiting autophagosome formation and reducing autophagy, with no effect on the cell cycle, apoptosis, or aging. Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in inflammation, autoimmunity and cancer, mainly via its E3 ubiquitin ligase activity. Mass spectrometry and immunoprecipitation assays showed that TRIM21 interacted with NCAPH and decreased the protein stability of NCAPH via ubiquitination at the K11 lysine residue. Structural domain mutation analysis revealed that TRIM21 combined with NCAPH through its PRY/SPRY and CC domains and accelerated the degradation of NCAPH through the RING domain. Furthermore, TRIM21 promoted autophagosome formation and reduced cell proliferation by inhibiting NCAPH expression and the downstream AKT/mTOR pathway in cervical cancer cells. Immunohistochemical staining revealed that the protein expression of TRIM21 was negatively correlated with that of NCAPH and positively correlated with that of beclin-1 in cervical cancer tissues. Therefore, we provide evidence for the role of the TRIM21-NCAPH axis in cervical cancer autophagy and proliferation and the involvement of the AKT/mTOR signaling pathway in this process. These results deepen our understanding of the carcinogenesis of cervical cancer, broaden the understanding of the molecular mechanisms of TRIM21 and NCAPH, and provide guidance for individualized treatment of cervical cancer in the future.

ATG10S promotes IFNL1 expression and autophagic degradation of multiple viral proteins mediated by IFNL1

ABSTRACT ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12–ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug. Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein

Stromal interaction molecule 1/microtubule-associated protein 1A/1B-light chain 3B complex induces metastasis of hepatocellular carcinoma by promoting autophagy.

Metastasis is the leading cause of death in hepatocellular carcinoma (HCC) patients, and autophagy plays a crucial role in this process by orchestrating epithelial-mesenchymal transition (EMT). Stromal interaction molecule 1 (STIM1), a central regulator of store-operated calcium entry (SOCE) in nonexcitable cells, is involved in the development and spread of HCC. However, the impact of STIM1 on autophagy regulation during HCC metastasis remains unclear. Here, we demonstrate that STIM1 is temporally regulated during autophagy-induced EMT in HCC cells, and knocking out (KO) STIM1 significantly reduces both autophagy and EMT. Interestingly, STIM1 enhances autophagy through both SOCE-dependent and independent pathways. Mechanistically, STIM1 directly interacts with microtubule-associated protein 1A/1B-light chain 3B (LC3B) to form a complex via the sterile-α motif (SAM) domain, which promotes autophagosome formation. Furthermore, deletion of the SAM domain of STIM1 abolishes its binding with LC3B, leading to a decrease in autophagy and EMT in HCC cells. These findings unveil a novel mechanism by which the STIM1/LC3B complex mediates autophagy and EMT in HCC cells, highlighting a potential target for preventing HCC metastasis.

TGFβ1 Secretion in Megakaryocytes Is Autophagy-Dependent and Its Inhibition Ameliorates Myelofibrosis in Mice

Megakaryocytes (MKs) are large, polyploid platelet precursor cells that primarily reside within the bone marrow. Recent advances revealed that in addition to platelet production, MKs contribute to immunity and maintenance of bone marrow homeostasis, raising the question of whether these functions are hijacked in disease. One hallmark of primary and secondary myelofibrosis (MF), subsets of myeloproliferative neoplasms, is the accumulation of aberrant, immature MKs in the bone marrow. These release pro-fibrotic cytokines such as transforming growth factor β1 (TGFβ1) and interleukin 1β (IL1β) into the bone marrow, thus inducing fibrotic alterations that ultimately cause pancytopenia - the major cause of mortality in patients with MF. While current treatment strategies in MF aim to prevent MK proliferation and correct the differentiation defects, a direct targeting of cytokine release from MKs has not yet been explored. Recently, unconventional secretion of TGFβ1 and IL1β via secretory autophagy, a secretion pathway driven by the fusion of autophagosomes with the plasma membrane, has been shown to occur in other cell types. We therefore hypothesize that MKs may preferentially utilize secretory autophagy for cytokine release and that inhibiting autophagosome formation might therefore slow fibrosis progression in vivo. Previous studies suggested that TGFβ1 is stored within specialized α-granules in MKs. To test this, we cultured murine MKs and determined TGFβ1 co-localization with different α-granule proteins by super-resolution confocal microscopy. However, while we did not identify any correlation between TGFβ1 and α-granules, we found that TGFβ1 strongly co-localized with the autophagy marker light chain (LC) 3B, supporting a potential role for secretory autophagy. To investigate this, we disrupted secretory autophagy by inhibiting the GTPase RhoA and its downstream effector Rho kinase (ROCK), which were previously shown to promote autophagosome formation. Treatment of MKs with inhibitors of autophagy or RhoA/ROCK markedly reduced TGFβ1 secretion in vitro and led to intracellular accumulation of TGFβ1. Taken together, these data suggest that TGFβ1 is secreted by MKs via secretory autophagy. To examine whether secretory autophagy in MKs contributes to disease pathogenesis in vivo, we employed an MPL W515L-driven transplant model. Basal autophagy measured by LC3B conversion and RhoA protein levels were increased in platelets derived from mice with MPL W515L-driven MF. Conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system ( Mx1-Cre+; Atg5fl/fl mice) prevented leukocytosis, MK clustering and aberrant TGFβ1 secretion in the MPL W515L-driven transplant model, indicating that autophagy promotes the initiation of MF. Furthermore, MPL W515L-transplanted mice treated with a low-dose ROCK inhibitor (ROCKi) - several of which are FDA-approved - reduced MK clustering and fibrosis in spleens and femurs of treated mice (Figure 1a, b). To further verify our data, we utilized a second preclinical mouse model of MF - a knockout mouse lacking the inhibitory MK receptor G6b-B ( Mpig6b -/-), which exhibits thrombocytopenia, MK clustering and MF. In line with our previous findings, conditional deletion of Rhoa in MK lineage cells in Pf4-Cre+; Rhoafl/fl; Mpig6b -/- mice normalized TGFβ1 levels within the bone marrow and protected against the development of fibrosis in bone marrow and spleen. In ongoing work, ROCK inhibitors are further being tested in higher doses alone or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data suggest that TGFβ1 secretion is uncoupled from conventional granule secretion in MKs and is instead dependent on RhoA/autophagy signaling pathways. Targeting these pathways in aberrant MKs represents a new potential therapeutic target in the treatment of MF.

Placental extract suppresses lipid droplet accumulation by autophagy during the differentiation of adipose-derived mesenchymal stromal/stem cells into mature adipocytes

ObjectivePlacental extract, which contains various bioactive compounds, has been used as traditional medicine. Many studies have demonstrated additional applications of placental extract and provided a scientific basis for the broad spectrum of its effects. We have previously reported that porcine placental extract (PPE) strongly suppresses adipogenesis in a 3T3-L1 preadipocyte cell line, inhibiting differentiation. This study aimed to examine the effect of PPE on the accumulation of lipid droplets (LD) in adipose-derived mesenchymal stromal/stem cells (ASC).ResultsThe study findings revealed that PPE decreased the size of LD during the differentiation of ASC into mature adipocytes. RT-qPCR analysis revealed that PPE increased the gene expression of lysosomal acid lipase A (Lipa), a lipolysis-related gene, in ASC-differentiated adipocytes. However, no differences were noted in the adipocyte differentiation markers (Pparg, Cebpa, and Adipoq), or the adipogenesis-related genes (Dgat1, Dgat2, Fasn, Soat1, and Soat2). In addition, PPE promoted autophagosome formation, which was partially co-localized with the LD, indicating that PPE accelerated the degradation of LD by inducing autophagy (termed lipophagy) during the differentiation of ASC into mature adipocytes. These results suggest that the use of PPE may be a potential novel treatment for regulating adipogenesis for the treatment of obesity.

Activation of mitophagy improves cognitive dysfunction in diabetic mice with recurrent non-severe hypoglycemia

Recurrent non-severe hypoglycemia (RH) in patients with diabetes might be associated with cognitive impairment. Previously, we found that mitochondrial dysfunction plays an important role in this pathological process; however, the mechanism remains unclear. The objective of this study was to determine the molecular mechanisms of mitochondrial damage associated with RH in diabetes mellitus (DM). We found that RH is associated with reduced hippocampal mitophagy in diabetic mice, mainly manifested by reduced autophagosome formation and impaired recognition of impaired mitochondria, mediated by the PINK1/Parkin pathway. The same impaired mitophagy initiation was observed in an in vitro high-glucose cultured astrocyte model with recurrent low-glucose interventions. Promoting autophagosome formation and activating PINK1/Parkin-mediated mitophagy protected mitochondrial function and cognitive function in mice. The results showed that impaired mitophagy is involved in the occurrence of mitochondrial dysfunction, mediating the neurological impairment associated with recurrent low glucose under high glucose conditions.

α-Hederin induces human colorectal cancer cells apoptosis through disturbing protein homeostasis

Protein homeostasis and quality control are crucial for normal cellular activities, and a severe imbalance in protein homeostasis can lead to cell death. α-Hederin, a pentacyclic triterpenoid saponin isolated from Fructus Akebia, has a clear role in promoting colorectal cancer (CRC) cell apoptosis and has been recently used for CRC therapy. However, whether the pro-apoptotic activity of α-hederin in CRC cells involves disturbing protein homeostasis remains unknown. Here, we aimed to uncover the underlying molecular mechanism of α-hederin-induced apoptosis in CRC cells. Cell viability and proliferation were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and 5-ethynyl-2′-deoxyuridine (EdU) assays, respectively. Apoptosis was detected using flow cytometry and western blotting. Autophagic flux was examined by western blotting and AdPlus-mCherry-GFP-LC3B adenovirus infection assays, and western blotting and immunofluorescence staining were performed to detect the expression of proteins in related pathways. The results showed that α-hederin significantly inhibited the growth and promoted the apoptosis of human CRC cells. Furthermore, α-Hederin induced endoplasmic reticulum (ER) stress, but inhibited proteasomal degradation. Also, the autophagic flux was blocked by α-hederin although this drug promoted autophagosome formation, and the lysosomal degradation was inhibited. Expression of p-JNK and p-p38 were increased by α-hederin. So, our findings provide strong evidence that α-hederin disrupts protein homeostasis by blocking ER-associated degradation (ERAD) and autophagic flux, thereby contributing to apoptosis. PERK-eIF2α-ATF4-CHOP and IRE1-ASK1-JNK/p38 signal pathway may be involved in those regulation. Our results make it a promising alternative or adjunct therapeutic candidate for CRC.

Chloroquine inhibited Helicobacter pylori-related gastric carcinogenesis by YAP–β-catenin–autophagy axis

YAP participates in autophagy associated with many diseases. In this study, we demonstrate that YAP promotes autophagy by interacting with beclin 1, upregulating beclin 1 and LC3B-II protein expression, and promoting autophagosome formation after H. pylori infection in a vacuolating cytotoxin A-dependent manner. The protein levels of β-catenin in the cytoplasm and nuclei of GES-1 cells and the mRNA levels of Axin2, Myc, Lgr5, and Ccnd1 were increased in H. pylori-infected cells or YAP-overexpressed cells, but were decreased in YAP-silenced cells. The β-catenin inhibitor XAV939 significantly downregulated autophagy, whereas the activator LiCl showed opposite effects. An H. pylori-infected mouse model of gastric carcinoma was successfully established. The mouse model showed that H. pylori infection, when combined with NMU, promoted the tumorigenesis of gastric tissues; increased IL-1β, IL-6, and TNF-α levels; promoted NO release; and increased the expression of beclin 1, LC3B-II more than NMU alone. Chloroquine inhibited these phenomena, but did not completely attenuate the effects of H. pylori. These results demonstrate that chloroquine can be used as a drug for the treatment of H. pylori-related gastric cancer, but the treatment should simultaneously remove H. pylori.

HD-sEVs in bovine follicular fluid regulate granulosa cell apoptosis and estradiol secretion through the autophagy pathway

Follicular fluid (FF) is rich in extracellular vesicles (EVs), which have regulatory effects on follicular growth and oocyte development. EVs can be divided into two subtypes, i.e. HD-sEVs and LD-sEVs. In this study, HD-sEVs were successfully isolated from bovine follicular fluid (BFF) by density gradient ultracentrifugation. By western blot, quantitative polymerase chain reaction (qPCR), flow cytometry, transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA), this study found HD-sEVs promoted autophagy in bGCs by increasing the protein and mRNA expression of LC3II/LC3I ratio and Beclin1, and inhibiting the protein and mRNA expression of p62. HD-sEVs promoted mitophagy in bGCs by increasing the protein and mRNA expression of VDAC1, CTSD, and HSP60. Flow cytometry showed that HD-sEVs inhibited bGCs apoptosis rate. HD-sEVs promoted estradiol secretion by increasing steroidogenesis-associated proteins and mRNA, such as CYP19A, HSD3B in bGCs. HD-sEVs promoted autophagosome formation and mitochondrial structure swelling in bGCs, and decreased p-mTOR/mTOR ratio. The above phenomenon was reversed when wortmannin was added. Collectively, BFF HD-sEVs promote bGCs autophagy and mitophagy, inhibit bGCs apoptosis and promote estradiol secretion through the autophagy pathway-mTOR signaling pathway.

Sinomenine improves renal fibrosis by regulating mesenchymal stem cell-derived exosomes and affecting autophagy levels.

This study attempts to investigate the therapeutic effect of sinomenine on renal fibrosis and its mechanism. The 8-week-old C57BL/6 male mice were randomly divided into sham group, UUO model group, UUO sinomenine group (UUO + Sino 50), UUO + sinomenine group (UUO + Sino 100), UUO + exosome group (exo), and UUO + exo-inhibitor. The pathological changes of kidney were observed by H&E staining, the degree of renal interstitial fibrosis was detected by MASSON and Sirius red staining, and the expressions of fibrosis and autophagy markers were detected by real-time fluorescence quantitative PCR and WB. NTA and electron microscopy were used to analyze exo secretion after sinomenine treatment. Sinomenine could improve the progression of renal fibrosis without causing tissue damage including heart, lungs and liver. Sinomenine could promote autophagosome formation. It could promote the secretion of exosomes from bone marrow mesenchymal stem cells (BMSCs). Sinomine regulates the PI3K-AKT pathway through BMSC-exo carrying miR-204-5p, affecting autophagy level and alleviating the process of renal fibrosis. Our study suggests that sinomine could improve the progression of renal fibrosis by influencing the expression of miR-204-5p in BMSC-exo and regulating the PI3K-AKT pathway.

WITHDRAWN: Transcription factor EB (TFEB) promotes autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) is responsible for devastating outcomes for patients with subarachnoid hemorrhage (SAH). Autophagy and apoptosis regulate the process of cell death. The transcription factor EB (TFEB) can increase autophagic flux by promoting autophagosome formation and autophagosome-lysosome fusion, and dysregulation of TFEB activity might induce the development of several diseases. However, the biological functions of TFEB in EBI post-SAH remain unknown. We established an animal model of SAH by modified endovascular perforation and a cellular model by treating primary cortical neurons with oxyhemoglobin (oxyHb). A recombinant adenovirus containing a shRNA targeting TFEB (adv-sh-TFEB) was used to knock down TFEB expression. Protein levels of TFEB, Bax, Bcl-2, cleaved caspase-3, LC3, and Beclin-1 were measured by western blotting and immunofluorescence staining. Neuronal apoptosis was assessed by flow cytometry and TUNEL staining. Short-term neurobehavioral functions were examined by modified Garcia and beam balance scores. Brain edema was determined through assessment of brain water content. TFEB was increased at the protein level in cellular and animal models of SAH. TFEB depletion attenuated the oxyHb-induced apoptosis of primary cortical neurons and autophagy. TFEB downregulation improved the short-term neurobehavioral functions and reduced brain edema after SAH and inhibited neuronal apoptosis in SAH rats. TFEB depletion improves short-term neurologic performance and reduces brain edema by preventing autophagy and apoptosis after SAH.

Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved self‑digestion process that is critical for maintaining homeostasis in response to various stresses. The autophagy‑related protein family, including the GABA type A receptor‑associated protein (GABARAP) and microtubule‑associated protein 1 light chain 3 subfamilies, is crucial for autophagosome biogenesis. Although the regulatory machinery of autophagy in the cytoplasm has been widely studied, its transcriptional and epigenetic regulatory mechanisms still require more targeted investigations. The present study identified histone lysine demethylase 3B (KDM3B) as a crucial component of autophagy on a panel of leukemia cell lines, including K562, THP1 and U937, resulting in transcriptional activation of the autophagy‑related gene GABA type A receptor‑associated protein like 1 (GABARAPL1). KDM3B expression promoted autophagosome formation and affected the autophagic flux in leukemia cells under the induction of external stimuli. Notably, RNA‑sequencing and reverse transcription‑quantitative PCR analysis showed that KDM3B knockout inhibited the expression of GABARAPL1. Chromatin immunoprecipitation‑quantitative PCR and luciferase assay showed that KDM3B was associated with the GABARAPL1 gene promoter under stimulation and enhanced its transcription. The present findings demonstrated that KDM3B was critical for regulating the GABARAPL1 gene and influencing the process of autophagy in leukemia cells. These results provide a new insight for exploring the association between autophagy and KDM3B epigenetic regulation in leukemia.

Dimethyl fumarate ameliorates parkinsonian pathology by modulating autophagy and apoptosis via Nrf2-TIGAR-LAMP2/Cathepsin D axis

Mounting evidence suggests a role for oxidative stress and accumulation of dysfunctional organelle and misfolded proteins in PD. Autophagosomes mediate the clearance of these cytoplasmic proteins via delivery to lysosomes to form autophagolysosomes, followed by degradation of the protein by lysosomal enzymes. In PD, autophagolysosome accumulation occurs initiating a plethora of events resulting in neuronal death by apoptosis. This study evaluated the effect of Dimethylfumarate (DMF), an Nrf2 activator in the rotenone-induced mouse PD model. In PD mice, there was decreased expression of LAMP2 and LC3, which resulted in inhibition of autophagic flux and increased expression of cathepsin D, which mediated apoptosis. The role of Nrf2 activation in alleviating oxidative stress is well known. Our study elucidated the novel mechanism underlying the neuroprotective effect of DMF. The loss of dopaminergic neurons induced by rotenone was lessened to a significant extent by pre-treatment with DMF. DMF promoted autophagosome formation and inhibited apoptosis by removing the inhibitory effect of p53 on TIGAR. TIGAR expression upregulated LAMP2 expression and downregulated Cathepsin D, promoting autophagy and inhibiting apoptosis. Thus, it was proved that DMF confers neuroprotection against rotenone-induced dopaminergic neurodegeneration and could be used as a potential therapeutic agent for PD and its progression.

Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson’s risk variant

ABSTRACT At the synapse, proteins are reused several times during neuronal activity, causing a decline in protein function over time. Although emerging evidence supports a role of autophagy in synaptic function, the precise molecular mechanisms linking neuronal activity, autophagy and synaptic dysfunction are vastly unknown. We show how extracellular calcium influx in the pre-synaptic terminal constitutes the initial stimulus for autophagosome formation in response to neuronal activity. This mechanism likely acts to rapidly support synaptic homeostasis and protein quality control when intense neuronal activity challenges the synaptic proteome. We identified a residue in the flexible region of EndoA (Endophilin A) that dictates calcium-dependent EndoA mobility from the plasma membrane to the cytosol, where this protein interacts with autophagic membranes to promote autophagosome formation. We discovered that a novel Parkinson’s disease-risk mutation in SH3GL2 (SH3 domain containing GRB2 like 2, endophilin A1) disrupts the calcium sensing of SH3GL2, leading to an immobile protein that cannot respond to calcium influx and therefore disrupting autophagy induction at synapses. Our work shows how neuronal activity is connected with autophagy to maintain synaptic homeostasis and survival.

Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.

Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.

Homoplantaginin attenuates high glucose-induced vascular endothelial cell apoptosis through promoting autophagy via the AMPK/TFEB pathway.

Vascular endothelial cell (VEC) injury is a key factor in the development of diabetic vascular complications. Homoplantaginin (Hom), one of the main flavonoids from Salvia plebeia R. Br. has been reported to protect VEC. However, its effects and mechanisms against diabetic vascular endothelium remain unclear. Here, the effect of Hom on VEC was assessed using high glucose (HG)-treated human umbilical vein endothelial cells and db/db mice. In vitro, Hom significantly inhibited apoptosis and promoted autophagosome formation and lysosomal function such as lysosomal membrane permeability and the expression of LAMP1 and cathepsin B. The antiapoptosis effect of Hom was reversed by autophagy inhibitor chloroquine phosphate or bafilomycin A1. Furthermore, Hom promoted gene expression and nuclear translocation of transcription factor EB (TFEB). TFEB gene knockdown attenuated the effect of Hom on upregulating lysosomal function and autophagy. Moreover, Hom activated adenosine monophosphate-dependent protein kinase (AMPK) and inhibited the phosphorylation of mTOR, p70S6K, and TFEB. These effects were attenuated by AMPK inhibitor Compound C. Molecular docking showed a good interaction between Hom and AMPK protein. Animal studies indicated that Hom effectively upregulated the protein expression of p-AMPK and TFEB, enhanced autophagy, reduced apoptosis, and alleviated vascular injury. These findings revealed that Hom ameliorated HG-mediated VEC apoptosis by enhancing autophagy via the AMPK/mTORC1/TFEB pathway.

RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer

Cancer-derived small extracellular vesicles (sEVs) serve as critical mediators of cell-to-cell communication. Manzamine A (MA), a unique marine-derived alkaloid with various bioactivities, exerts anticancer effects against several kinds of tumors, but it remains unclear whether it has the same activity against breast cancer. Here, we proved that MA inhibits MDA-MB-231 and MCF-7 cell proliferation, migration, and invasion in a time- and dose-dependent manner. In addition, MA promotes autophagosome formation but suppresses autophagosome degradation in breast cancer cells. Importantly, we also found that MA stimulates sEVs secretion and increases autophagy-related protein accumulation in secreted sEVs, further potentiated by autophagy inhibitor chloroquine (CQ). Mechanistically, MA decreases the expression level of RIP1, the key upstream regulator of the autophagic pathway, and reduces the acidity of lysosome. Overexpression of RIP1 activated AKT/mTOR signaling, thus attenuating MA-induced autophagy and the corresponding secretion of autophagy-associated sEVs. Collectively, these data suggested that MA is a potential inhibitor of autophagy by preventing autophagosome turnover, and RIP1 mediates MA-induced secretory autophagy, which may be efficacious for breast cancer treatment.

Discovery of novel 2-oximino-2-indolylacetamide derivatives as potent anticancer agents capable of inducing cell autophagy and ferroptosis

A series of 2-oximino-2-indolylacetamide derivatives were designed, synthesized and evaluated for their antitumour effects. Among them, 4d exhibited the most potent antiproliferative effect in vitro on the tested human cancer cells. Additionally, 4d significantly induced cell apoptosis, caused mitochondrial dysfunction, promoted Bax, cleaved-PARP and p53 expression and inhibited Bcl-2 expression in 5-8F cells. Moreover, 4d remarkably promoted autophagosome formation, leading to cell apoptosis. Further investigation indicated that 4d could trigger cell death through cell ferroptosis, including increased ROS generation and lipid peroxidation and decreased glutathione peroxidase 4 (GPx4) expression and glutathione (GSH) levels. More importantly, 4d induced 5-8F cell death by activating ROS/MAPK and inhibiting the AKT/mTOR and STAT3 signalling pathways. Interestingly, 4d significantly suppressed tumour growth in a 5-8F cell xenograft model without obvious toxicity to mice. Overall, these results demonstrate that 4d may be a potential compound for cancer therapy.

The COPS3-FOXO3 positive feedback loop regulates autophagy to promote cisplatin resistance in osteosarcoma

ABSTRACT Chemotherapy is an important treatment modality for osteosarcoma (OS), but the development of chemoresistance limits the therapeutic efficacy of OS and results in a poor prognosis. Thus, a better understanding of the mechanisms underlying chemoresistance in OS is essential. We previously demonstrated that COPS3/CSN3 (COP9 signalosome subunit 3) functions as an oncogene to promote OS cells lung metastasis, which is closely related to chemoresistance. Here, we showed that COPS3 was significantly upregulated in OS tissues with poor response to preoperative chemotherapy. Moreover, COPS3 depletion made OS cells more sensitive to cisplatin treatment in vitro and in vivo, implicating COPS3 as a driver of cisplatin resistance. Mechanistic investigations showed that COPS3 induced a cytoprotective macroautophagy/autophagy in response to cisplatin. Specifically, we identified FOXO3 as a critical target of COPS3, as high expression of COPS3 enhanced the nuclear abundance of FOXO3 and increased the expression of FOXO3-responsive genes, promoting autophagosome formation and maturation. In turn, FOXO3 regulated COPS3 levels by inhibiting ubiquitin-mediated degradation and attenuating SKP2-mediated COPS3 inhibition, cooperatively maintaining a high level of COPS3. In both COPS3-expressing OS cells and a murine xenograft model, inhibition of autophagy could also overcome resistance to cisplatin. Collectively, our results offer insights into the mechanisms of cisplatin resistance and suggest that targeting COPS3-mediated autophagy is a promising therapeutic strategy for overcoming the cisplatin resistance of OS. Abbreviations: 3-MA: 3-methyladenine; BECN1: beclin 1; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; COPS3/CSN3: COP9 signalosome subunit 3; CQ: chloroquine; DEGs: differentially expressed genes; FOXO3: forkhead box O3; GFP: green fluorescent protein; IC50: 50% inhibitory concentration; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; mRFP: monomeric red fluorescent protein; OS: osteosarcoma; PBS: phosphate-buffered saline; qRT-PCR: quantitative real-time PCR; RAB7: RAB7, member RAS oncogene family; RPS6KB1/p70S6K1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; shRNA: short hairpin RNA; siRNA: small interfering RNA; SKP2: S-phase kinase associated protein 2; TEM: transmission electron microscopy; UPS: ubiquitin-proteasome system