ABSTRACT Enteroviruses pose a substantial global health burden, and a complete understanding of host defense mechanisms is still evolving. Through proteomic profiling of coxsackievirus B3 (CVB3)-infected cells, we identified the host factor USF2 (upstream transcription factor 2) as significantly upregulated, a process that may be associated with the viral capsid protein VP4. Here, we characterize USF2 as a novel, infection-induced restriction factor. Functional studies demonstrate that USF2 knockdown enhances viral RNA replication without affecting entry, while its overexpression suppresses replication and cytopathic effects. Mechanistically, USF2 acts as a transcriptional repressor of autophagy-related genes, thereby inhibiting autophagosome formation. Crucially, USF2 depletion promotes the release of MAP1LC3/LC3-positive extracellular vesicles carrying infectious virus, defining its role in blocking a key route of vesicle-mediated viral dissemination. USF2 exhibits broad antiviral activity against multiple enteroviruses and other RNA viruses that depend on cytoplasmic membrane remodeling. Our findings reveal that the host deploys a virus-triggered transcriptional regulator to restrict enterovirus spread by suppressing macroautophagy/autophagy-dependent viral dissemination.

ABSTRACT Macroautophagy/autophagy represents a promising therapeutic target in oncology, exhibiting context-dependent roles in tumor progression. Kinase inhibitors are a large group of anti-cancer drugs, and elucidating the regulatory effects of different kinase inhibitors on autophagy offers a valuable strategy to advance our understanding of autophagy regulation in cancer and develop innovative anti-cancer therapies. In this study, we developed a drug screening platform and performed high-content screening using a kinase inhibitor library. The screening identified PTK2/FAK (protein tyrosine kinase 2) inhibitors as potent autophagy inducers. Mechanistic investigations revealed that PTK2/FAK inhibition triggers rapid autophagy flux through a non-classical mechanism dependent on PTK2/FAK expression, which is associated with the spatial redistribution of ER exit sites (ERES). Further analysis demonstrated that PTK2/FAK inhibitor (FAKi)-induced ERES-associated autophagy is not contingent upon full PREB/SEC12 and SEC16 expression, but is sensitized to TMED9 expression level. Additionally, we identified AKAP13 as a novel FAKi-responsive protein that undergoes dephosphorylation upon FAKi treatment and contributes to TMED9-mediated ERES-associated autophagy. Given that ERES-associated autophagy is initiated through membrane contact between ERES and the ER-Golgi intermediate compartment (ERGIC), we further observed enhanced TMED9-ERGIC interactions following FAKi treatment. Furthermore, functional studies confirmed that FAKi-induced autophagy promotes pancreatic ductal adenocarcinoma (PDAC) cell survival both in vitro and in vivo. Collectively, our work unveils a previously unrecognized mechanism of FAKi-mediated autophagy induction and provides new insights for developing targeted therapies against PDAC.

Age-related macular degeneration (AMD) involves sub-retinal pigment epithelium (sub-RPE) lipid deposition in the early stage, with dysregulated lipid metabolism and impaired macroautophagy/autophagy implicated, yet the molecular mechanisms underlying their interaction remain unclear. In this study, transcriptomic analysis of human macular tissues identified FASN (fatty acid synthase), a regulator of lipid metabolism and lysosomal function, as a significantly upregulated key hub gene in early AMD. In apoe-/- mice fed a high-fat diet (HFD), retina-RPE-choroid complexes revealed elevated FASN alongside autophagy suppression, lysosomal dysfunction, and lipid accumulation. In vitro, FASN protein levels increased in RPE cells treated with the autophagy inhibitor 3-methyladenine (3-MA), but decreased with the autophagy activator rapamycin (RAPA), without transcriptional changes; lysosomal blockade with chloroquine (CQ) induced FASN accumulation, which was significantly delayed following autophagy inhibition. These findings indicate that FASN accumulation results from insufficient autophagic degradation. Conversely, FASN knockdown or pharmacological inhibition enhanced autophagic flux and promoted lysosomal lipid clearance in RPE cells. Mechanistically, FASN inhibition increased AMPK phosphorylation and decreased MTOR activity, thereby facilitating autophagy and lipophagy. Collectively, our findings reveal a self-amplifying pathological circuit in early AMD: autophagy impairment drives FASN accumulation, which in turn exacerbates lysosomal dysfunction and lipid accumulation. Targeting the FASN-AMPK-MTOR axis may offer a promising therapeutic strategy for early AMD.

ABSTRACT Glioblastoma (GB) is the most aggressive brain cancer, with poor prognosis due to infiltrative invasion of glioma stem cells (GSCs) and the immunosuppressive tumor microenvironment (TME). We have previously demonstrated that pericytes (PCs), specialized cells in the blood microvessels surrounding GB, are conditioned by infiltrating tumor cells to aberrantly upregulate their chaperone-mediated autophagy (CMA). Elevated CMA in PCs promotes stable cell-cell interactions with tumor cells and a pro-tumoral immune phenotype that supports tumor progression. In this work, to test if inhibition of CMA in PCs might be an effective strategy to reduce tumor survival, we have used the phosphopeptide P140, known to restore aberrant CMA upregulation in specific immune cells. We found that administration of P140 peptide in an immunocompetent GB mouse model with both patient-derived GSCs and GB cell lines, neutralizes GB-induced PC CMA upregulation resulting in ablation of PC-tumor cell interactions and triggering of a secretome toxic to tumor cells. We identified MAPT/tau, a known CMA substrate, as one of the main components of this secretome, and discovered that CMA-dependent PC MAPT/tau secretion within the GB TME plays a key role in cancer progression and recurrence. Furthermore, we found that perivascular accumulation of MAPT/tau is an effective way to monitor peptide P140 treatment efficacy in GB and for prognosis of the patient evolution. Our findings validate P140 peptide treatment as a safe and specific strategy to halt GB progression and establish the clinical relevance of extracellular MAPT/tau as a biomarker for therapeutic success in GB. Abbreviations: ACTA2/ASMA: actin alpha 2, smooth muscle; BBB: blood-brain barrier; CMA: chaperone-mediated autophagy; CTLA4: cytotoxic T-lymphocyte associated protein 4; DAB: 3–3′diaminobenzidine; GB: glioblastoma; GBCPC: GB-conditioned pericyte; GSCs: glioblastoma stem cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosome associated membrane protein 2A; LI: lysosomal inhibitors; MAPT/tau: microtubule associated protein tau; NAc: N-acetylcysteine; PC: pericyte; PCGB: pericyte-glioblastoma coculture; PDGFRB: platelet derived growth factor receptor beta; ROS: Reactive oxygen species; SPARC: secreted protein acidic and cysteine rich; TME: tumor microenvironment; TNT: tunneling nanotubes

ABSTRACT Ryanodine receptors (RYRs) are ER-resident Ca2+-release channels enriched in excitable cells, including neurons. RYR hyperactivity is implicated in early pathogenesis of disorders such as Alzheimer’s disease (AD), which is associated with impaired autophagy. We recently uncovered a mechanism linking RYR activity to lysosome availability for autophagy. RYRs localize to ER-lysosome contact sites via direct binding to ATP6V0A1, a V-ATPase subunit that also suppresses RYR-mediated Ca2+ release. In human iPSC-derived cortical neurons, spontaneous RYR activity promotes lysosomal secretion, depleting the intracellular lysosomal pool and inhibiting autophagic flux. RYR inhibition promotes ERlysosome contacts, limits lysosomal secretion, and restores lysosome availability for autophagosome fusion and cargo degradation (including APP). Conversely, disrupting the RYR:ATP6V0A1 interaction using a RYR-derived protein fragment serving as a “decoy” for ATP6V0A1 evokes RYR hyperactivity and stimulates lysosomal secretion. In this Punctum, we discuss how this RYR2:ATP6V0A1 “contact-site hub” may be perturbed in disease and highlight open questions on how lysosomes decode RYR-derived Ca2+ signals.

ABSTRACT Selective autophagy of the endoplasmic reticulum (ER), termed ERphagy or reticulophagy, plays a key role in organelle remodeling and cellular homeostasis. However, whether and how ERphagy is regulated during Gram-negative bacteria infection to influence host responses remains unclear. Here, we show that Salmonella enterica serovar Typhimurium releases lipopolysaccharide (LPS) that colocalizes with RETREG1/FAM134B, a reticulon-like ER-resident receptor for ERphagy. Cytosolic delivery of LPS, either during infection or via transfection, markedly increases RETREG1- and LC3B-decorated ER fragments. Mechanistically, affinity-isolation assays demonstrate that LPS directly binds RETREG1 through interactions between lipid A and positively charged residues within its amphipathic helices and C-terminal region. This interaction promotes RETREG1 oligomerization and drives ER membrane fragmentation, a process further amplified by the O-antigen moiety of LPS. The resulting ER fragments accumulate around LC3-positive Salmonella-containing vacuoles, facilitating bacterial clearance. Importantly, both intracellular and extracellular Salmonella exploit outer membrane vesicles (OMVs) to deliver LPS into the host cytosol, triggering RETREG1 activation and ER remodeling. Collectively, our findings reveal a previously unrecognized host response by which LPS of Gram-negative bacteria are sensed by the host ERphagy machinery to promote xenophagy and enhance antibacterial defense. Abbreviations: AH: amphipathic helix; BMDMs: bone-marrow-derived macrophages; Co-IP: co-immunoprecipitation; BafA1: bafilomycin A1; Cterm: C-terminal region (Cterm); CFU: colony-forming units; DAPI: 4’,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; EPEC: enteropathogenic Escherichia coli; GBP: guanylate binding protein; Gm12250/IRGB10: predicted gene 12250; KDO: keto-3-deoxy-octonate; LPR: lipid-to-protein ratio; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mtLIR: LC3B-interacting region mutant; MDP: muramyl dipeptide; OMVs: outer membrane vesicles; O-Ag: O-antigen; OmpA: outer membrane protein A; RHD: reticulum homology domain; R-LPS: rough-LPS; S-LPS: smooth-LPS; SCVs: Salmonella-containing vacuoles; SFB: S-protein-FLAG-streptavidin binding peptide; TM: transmembrane domain; TEM: transmission electron microscopy; WT: wild-type.

ABSTRACT Macroautophagy/autophagy is a process conserved across eukaryotes that maintains cellular homeostasis by delivering cellular components to the vacuole or lysosome for further breakdown and recycling. Although the molecular mechanisms regulating autophagosome formation have been extensively studied, those underlying the modulation of autophagic body degradation in plant cells remain unclear. Here, we determined that VAB1 (V-ATPase catalytic subunit B1), a direct target of SINAT (SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA), is involved in the modulation of autophagic body degradation in plants. SINAT physically associated with VAB1 in vitro and in vivo to regulate its ubiquitination and degradation in planta. Compared with the wild-type (WT), the vab1 mutants showed decreased tolerance to nutrient starvation and premature leaf senescence. Moreover, deletion of vab1 led to impaired starvation-induced autophagic vesicle degradation by disrupting V-ATPase activity mediated vacuolar acidification. Consistently, lysines K34 and K221 of VAB1 protein contributed to SINAT1-mediated ubiquitination, destabilization, and suppression of autophagy-associated nutrient starvation tolerance. Thus, our findings demonstrate that SINAT proteins are involved in autophagy regulation by modulating VAB1-mediated autophagic degradation in Arabidopsis.

ABSTRACT Di(2-ethylhexyl) phthalate (DEHP) is a widely used industrial plasticizer, raising global concerns due to its potential endocrine-disrupting effects and environmental persistence. Human exposure to DEHP primarily occurs through the ingestion of contaminated food and water, inhalation of airborne particles, and dermal contact with products containing DEHP. Understanding the toxicological mechanisms of DEHP is essential for evaluating its health risks and developing effective strategies to mitigate its adverse effects. In this study, we conducted long-term exposure experiments to DEHP using both an animal model and in vitro system to investigate the complex interplay among DNA methylation, hyperactivation of macroautophagy/autophagy, mitochondrial dysfunction, and lipid accumulation induced by DEHP. The results revealed that DEHP exposure induced the degradation of DNMT1 (DNA methyltransferase 1) by enhancing its interaction with the autophagy-related protein SQSTM1 (sequestosome 1). DNMT1 degradation resulted in decreased methylation of the promoter regions of genes associated with autophagosome formation, subsequently increasing their expression. The resulting demethylation excessively activated autophagy, contributing to mitochondrial dysfunction and lipid accumulation in the liver. This study uncovered a previously unrecognized interplay among hyperactivation of autophagy, mitochondrial dysfunction, and lipid accumulation in the context of DEHP exposure. These findings enhanced our understanding of DEHP’s toxicity and underscored concerns about the long-term health effects of environmental pollutants, particularly regarding metabolic diseases. Abbreviation: ATG5:autophagy related 5; ATG16L1: autophagy related 16 like 1; BECN1:beclin 1; COX4/COXIV: cytochrome c oxidase subunit 4; BS-seq:bisulfite sequencing; DCFH-DA: 2′,7′-dichlorodihydrofluoresceindiacetate; DEHP: di(2-ethylhexyl) phthalate; DNMT1: DNAmethyltransferase 1; DNMT3A: DNA methyltransferase 3A; FABP4: fattyacid binding protein 4; FASN: fatty acid synthase; LPL: lipoproteinlipase; MAP1LC3/LC3: microtubule associated protein1 light chain 3; NAFLD: nonalcoholic fatty liver disease; NR1H3:nuclear receptor subfamily 1 group H member 3; PPARG: peroxisomeproliferator activated receptor gamma; RB1CC1: RB1 induciblecoiled-coil 1; SQSTM1: sequestosome 1; SREBF2: sterol regulatoryelement binding transcription factor 2; VDAC1: voltage dependentanion channel 1.

ABSTRACT DHODH (dihydroorotate dehydrogenase (quinone)) has been demonstrated as a critical regulator of programmed cell death, yet its role in macroautophagy/autophagy remains poorly defined. Flaviviridae pose a significant threat to global public health, and their replication is closely associated with autophagy. Building upon our previous findings that DHODH was a broad-spectrum target for Flaviviridae and a key regulator of Pestiviruses replication, this study employed RNA-seq screening coupled with functional validation to demonstrate that DHODH affected Pestiviruses replication by regulating mitophagy. Notably, we observed remarkable virus genus specificity in this regulatory mechanism. For autophagy-dependent Pestiviruses, DHODH deficiency impaired autophagosome-lysosome fusion, thereby suppressing viral replication. Conversely, in autophagy-inhibiting Flaviviruses, the blockade of autophagy flux facilitated viral replication. These observations underscore the specificity of DHODH-mediated viral replication regulation. Additionally, compound supplementation assays indicated that DHODH regulated autophagy via pyrimidine nucleotide metabolism, as exogenous pyrimidine precursors restored autophagosome-lysosome fusion. Furthermore, our research uncovered a novel mechanism whereby classical swine fever virus (CSFV) non-structural protein 4A (NS4A) recruited DHODH to mitochondria, facilitating its interaction with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) through the LC3-interacting region (LIR) domain to activate mitophagy. Collectively, our findings highlight DHODH as a promising antiviral target within the metabolism-autophagy axis, providing novel insights for antiviral drug development. Abbreviation: AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; ATG5: autophagy related 5; BafA1: bafilomycin A1; BNIP3L/NIX: BCL2 interacting protein 3 like; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; co-IP: co-immunoprecipitation; COX4: cytochrome c oxidase subunit 4; CQ: chloroquine; CSFV: classical swine fever virus; DAPI: 4’,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DHO: DHODH substrate dihydroorotate; DHODH: dihydroorotate dehydrogenase; DTMUV: duck tembusu virus; FIS1: fission mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HSPA/HSP70: heat shock protein family A (Hsp70); JEV: Japanese encephalitis virus; KEGG: kyoto encyclopedia of genes and genomes; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mdivi-1: mitochondrial division inhibitor 1; MFF: mitochondrial fission factor; MFN1: mitofusin 1; MFN2: mitofusin 2; MITO: mitochondria; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; MTS: mitochondrial targeting signal; OPTN: optineurin; ORO: DHODH product orotate; PBS: phosphate-buffered saline; PRKN: parkin RBR E3 ubiquitin protein ligase; PYR: pyrazofurin; RAPA: rapamycin; RFP: red fluorescent protein; RNA-seq: RNA sequencing; RT-qPCR: reverse transcription-quantitative real-time polymerase chain reaction; SD: standard deviation; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; UMP: uridine monophosphate; VDAC1: voltage dependent anion channel 1

ABSTRACT The CGAS-STING1 pathway plays a key role in detecting cytosolic DNA and initiating immune responses. Excessive STING1 activation can lead to aberrant inflammation and autoinflammatory diseases; therefore, the STING1 degradation pathway is tightly regulated by several negative regulatory mechanisms. In our recent study, we show that the selective autophagy receptor TAX1BP1 functions as a negative regulator of STING1 signaling. TAX1BP1 promotes the degradation of activated STING1 through microautophagy by facilitating the interaction of STING1 with the ESCRT-0 protein HGS, and selective autophagy of the Golgi apparatus in a process known as Golgiphagy. In TAX1BP1-deficient macrophages, STING1 aggregates accumulate at the trans-Golgi network, leading to stronger antiviral and inflammatory responses. These findings support a novel mechanism linking organelle quality control and innate immune regulation, highlighting Golgiphagy as an important feedback mechanism that limits STING1 signaling. Abbreviations: cGAMP: cyclic guanosine monophosphate-adenosine monophosphate; CGAS: Cyclic GMP-AMP synthase; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; ECTV: ectromelia virus; HGS: hepatocyte growth factor-regulated tyrosine kinase substrate; IKK: IκB kinase; IRF3: interferon regulatory factor 3; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TAX1BP1: Tax1 binding protein 1.