Autophagy最新文献

Lysosome homeostasis is vital for cellular fitness due to the essential roles of this organelle in various pathways. Given their extensive workload, lysosomes are prone to damage, which can stimulate lysosomal quality control mechanisms such as biogenesis, repair, or autophagic removal - a process termed lysophagy. Despite recent advances highlighting lysophagy as a critical mechanism for lysosome maintenance, the extent of lysosome integrity perturbation and the magnitude of lysophagy in vivo remain largely unexplored. Additionally, the pathophysiological relevance of lysophagy is poorly understood. To address these gaps, it is necessary to develop quantifiable methods for evaluating lysosome damage and lysophagy flux in vivo. To this end, we created two transgenic mouse lines expressing a tandem fluorescent LGALS3/galectin 3 probe (tfGAL3), either constitutively or conditionally under Cre recombinase control, utilizing the property of LGALS3 to recognize damaged lysosomes. This tool enables spatiotemporal visualization of lysosome damage and lysophagy activity at single-cell resolution in vivo. Systemic analysis across various organs, tissues, and primary cultures from these lysophagy reporter mice revealed significant variations in basal lysophagy, both in vivo and in vitro. Additionally, this study identified substantial changes in lysosome integrity and lysophagy flux in different tissues under stress conditions such as starvation, acute kidney injury and diabetic modeling. In conclusion, these complementary lysophagy reporter models are valuable resources for both basic and translational research.Abbreviation: AAV: adeno-associated virus; ATG7: autophagy related 7; CA-tfGAL3: cre-recombinase-activated tandem fluorescent LGALS3; DAPI: 4',6-diamidino-2-phenylindole; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HFD: high-fat diet; Igs2/H11/Hipp11: intergenic site 2; IST1: IST1 factor associated with ESCRT-III; KI: knock-in; LAMP1: lysosomal-associated membrane protein 1; LGALS3: lectin, galactoside-binding, soluble, 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; MEFs: mouse embryonic fibroblasts; NaOx: sodium oxalate; PDCD6IP: programmed cell death 6 interacting protein; PTECs: proximal tubular epithelial cells; RFP: red fluorescent protein; STZ: streptozotocin; TAM: tamoxifen; tfGAL3: tandem fluorescent LGALS3; TMEM192: transmembrane protein 192.

Macroautophagy/autophagy has long been viewed as being strictly dependent on vacuolar or lysosomal acidity, with the vacuolar-type H⁺-translocating ATPase (V-ATPase) functioning mainly as a proton pump that sustains degradation. Our recent paper overturns this paradigm, revealing that loss of V-ATPase activity paradoxically induces a selective autophagy program in nutrient-replete Saccharomyces cerevisiae. Vacuolar deacidification triggers a signaling cascade through the Gcn2-Gcn4/ATF4 integrated stress response, which drives Atg11-dependent ribophagy even when TORC1 remains active. This "V-ATPase-dependent autophagy" operates as a self-corrective feedback loop: when the vacuole's degradative capacity falters, it signals its own dysfunction to restore homeostasis. Tryptophan and NAD⁺ metabolism modulate this response, linking metabolic balance to autophagy induction. This discovery reframes the vacuole/lysosome from a passive endpoint to an active sensor of cellular integrity. It also challenges the use of V-ATPase inhibitors such as bafilomycin A₁ as neutral autophagy flux blockers, because inhibition itself can stimulate autophagy induction. Collectively, these findings position the V-ATPase as a bidirectional regulator - both gatekeeper and sentinel - governing how cells translate organelle stress into adaptive autophagy.Abbreviation: ATG: autophagy related; FL: follicular lymphoma; TORC1: TOR complex 1; V-ATPase: vacuolar-type H⁺-translocating ATPase.

Members of the mammalian Atg8-protein family (ATG8), including the MAP1LC3/LC3 and GABARAP subfamilies, play essential roles in selective macroautophagy/autophagy. However, their functional distinctions during viral infection remain poorly understood. Here, we show that S-adenosyl-L-methionine (SAM)-binding viral proteins, such as nsp14 from coronavirus and NP868R from African swine fever virus (ASFV), reprogram autophagy by shifting antiviral LC3B activity toward GABARAP-mediated mitophagy in an ATG4A-dependent manner. Mechanistically, the SAM-binding motif allows these viral proteins to stabilize ATG4A mRNA, thereby increasing ATG4A expression and redirecting autophagic flux from LC3B-mediated virophagy to GABARAP-dependent mitophagy. This shift suppresses innate immune responses by targeting both MAVS-dependent interferon signaling and virophagy, ultimately enhancing viral replication. Collectively, our findings uncover a previously unrecognized immune evasion strategy in which SAM-binding viral proteins rewire autophagy from antiviral to proviral pathways.Abbreviation: ACTB: actin beta; ATG: autophagy related genes; ASFV: African swine fever virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CS: citrate synthase; ExoN: exoribonuclease; GABARAP: GABA type A receptor-associated protein; IFN: type I interferon; IFNB: interferon beta; IPEC-J2: intestinal porcine epithelial cell line-J2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; nsp14: nonstructural protein 14; OPTN: optineurin; PEDV: porcine epidemic diarrhea virus; RNMT/N7-MTases: RNA guanine-7 methyltransferase; SAM: S-adenosyl-L-methionine; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TCID₅₀: 50% tissue culture infective dose; TOMM70: translocase of outer mitochondrial membrane 70; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type.

Intrahepatic triglyceride breakdown and recycling occur through lipolysis and lipid droplet (LD) macroautophagy/autophagy to regulate systemic fat partitioning. We recently demonstrated that MC3R is important for hepatic autophagy and peripheral metabolism, beyond its established functions in the CNS, where it affects energy homeostasis, feeding regulation, and puberty. MC3R agonists activate hepatocyte autophagy through LC3-II activation, TFEB cytoplasmic-to-nuclear translocation, and subsequent downstream autophagy gene activation. Global mc3r knockout mice develop obesity with increased hepatic triglyceride accumulation and blunted hepatocellular autophagosome-lysosome docking, leading to defective lipid droplet clearance. Hepatic Mc3r reactivation in global knockouts improves hepatocellular autophagy, lipid metabolism, mitochondrial respiration, energy expenditure, body fat, and body weight. These results reveal an autonomous role for hepatic MC3R in regulating lipid droplet autophagy, liver steatosis, and systemic adiposity.Abbreviation: AP:autophagosome; CNS:central nervous system; EIF4EBP1: eukaryotic translation initiationfactor 4E binding protein 1; EM: electron microscopy; LD: lipiddroplets; GFP: green fluorescent protein; MAFLD: metabolic-associatedfatty liver disease; MAP1LC3/LC3-II: microtubule-associated protein 1light chain 3-II; MC3R: melanocortin 3 receptor; MTORC1: mechanistictarget of rapamycin kinase complex 1; NDP-42:norleucine D-phenylalanine compound-42; TFEB: transcriptionfactor EB.

Macroautophagy/autophagy is a highly conserved catabolic membrane trafficking process through which various intracellular constituents, from proteins to organelles, are targeted for vacuolar/lysosomal degradation. Autophagy is tightly regulated both temporally and in magnitude at multiple levels to prevent either excessive or insufficient activity. To date, only a few RNA-binding proteins have been characterized as regulating the expression of genes essential for autophagy, and the contribution of post-transcriptional regulation in autophagy activity remains poorly understood. Here, through a genetic screen for autophagy-defective mutants, we identified Npl3, a nucleus-cytoplasm shuttling mRNA-binding protein, as essential for both bulk and selective types of autophagy. Deletion of NPL3 does not affect autophagosome biogenesis, closure, or maturation; however, it severely impairs autophagosome-vacuole fusion and results in minimal autophagosome turnover. We further demonstrated that this regulation depends on the RNA-binding domain of Npl3 and its capability for nuclear re-import. Together, our results reveal a novel layer of post-transcriptional regulation of autophagy.Abbreviations: Atg,autophagy related; HOPS: homotypic fusion and protein sorting; prApe1: precursor aminopeptidase I; RBP, RNA-binding protein; RRM, RNA-recognition motif; SNARE: soluble NSF attachment protein receptor; PAS: phagophore asse.

Antigen presentation by major histocompatibility complex class I (MHC class I) molecules is crucial for activating the T-cell-mediated immune response. A recent paper by Herhaus and colleagues revealed that IRGQ (immunity related GTPase Q) functions as an autophagy receptor for MHC class I molecules. IRGQ, being an oncogenic macroautophagy/autophagy receptor, also functions as an immune modulator, thus presenting a novel functional example. IRGQ regulates the quality control of MHC class I molecules, thereby influencing the T-cell-mediated immune response; IRGQ directs misfolded MHC class I molecules to autophagic degradation, thereby suppressing the immune response and mediating tumor evasion. Conversely, in the absence of IRGQ, free MHC class I heavy chains can reach the cell surface, potentially enhancing the immune response and suppressing tumor evasion. The results describe a novel example of autophagy promoting tumor evasion through immunomodulation. Indeed, the study found that lower levels of IRGQ are associated with higher survival rates in patients with hepatocellular carcinoma (HCC) and in a mouse model of HCC.

Macroautophagy/autophagy is best known for its role in maintaining cellular homeostasis through degradation of damaged proteins and organelles. In neurons, autophagy also contributes to the regulation of activity by adjusting the availability of cellular components to physiological demand. In a recent study, we show that autophagy shapes neuronal excitability by restraining a calcium-dependent pathway that couples endoplasmic reticulum calcium release to KCNMA1/BKCa activity at the plasma membrane. When autophagy is lost, this pathway is enhanced, and seizure susceptibility increases.

The inorganic pyrophosphatase PPA2, a matrix-localized protein, maintains mitochondrial function. Here, we identified the role of PPA2 in activating mitochondrial fission signaling. We found that PPA2 overexpression promotes mitochondrial fission by upregulating the mitochondrial translocation of phosphorylated DNM1L S616. Moreover, PPA2 interacts with MTFP1, a mitochondrial inner membrane protein, to induce fission signaling; cells knocked down for MTFP1 and overexpressing PPA2 failed to induce DNM1L activation and subsequent mitochondrial fission. Furthermore, in physiological conditions, PPA2 directed mitochondrial fission at the midzone through MFF-DNM1L, leading to mitochondrial proliferation. Interestingly, during mitochondrial stress following CCCP treatment, PPA2 triggers peripheral fission through FIS1 and DNM1L to segregate parts of damaged mitochondria, which is essential for mitophagy. In addition, PPA2 utilized the C-terminal LC3-interacting region (LIR) of MTFP1 for mitophagy-mediated clearance of damaged mitochondria. In conclusion, PPA2 activates mitochondrial fission signaling through MTFP1-DNM1L and is essential in defining the site of mitochondrial fission, leading to mitochondrial proliferation or mitophagy for maintaining mitochondrial homeostasis.Abbreviations: CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Co-IP: co-immunoprecipitation; CQ: chloroquine; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MLS: mitochondrial localization signal; mtDNA: mitochondrial DNA; OMM: outer mitochondrial membrane; RT: room temperature.

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are two major pathways for maintaining cellular protein homeostasis. Increasing evidence has highlighted the complex interactions and crosstalk between these pathways; however, the specific molecules and mechanisms mediating the interplay between the UPS and autophagy are still not fully elucidated. In this study, we discovered that knocking down the Drosophila Cul2 (Cullin 2)-RING ubiquitin ligase complex adaptor CG12084/DmZer1 impedes autophagy and autophagic flux. DmZer1 interacts with the Drosophila SQSTM1/p62 homolog ref(2)P, promoting its association with ubiquitinated proteins and degradation. ref(2)P is a crucial player in regulating autophagy and the Keap1-cnc/NFE2L2 pathway-mediated antioxidant response. Knockdown of DmZer1 leads to the formation of ref(2)P bodies, which sequester Keap1 and promote cnc/NFE2L2-mediated antioxidant responses under oxidative stress conditions. These findings reveal the pivotal role of DmZer1 in regulating autophagy and the ref(2)P-Keap1-cnc/NFE2L2-mediated oxidative stress response.Abbreviations: ARM: armadillo-like domain; ATG: autophagy related; BTZ: bortezomib; CL1-GFP: GFP fused with a CL1 degron; cnc: cap-n-collar; co-IP: co-immunoprecipitation; CRL2: Cullin 2-RING E3 ubiquitin ligase complex; CQ: chloroquine; CUL2/Cul2: cullin 2; EloB: Elongin B; EloC: Elongin C; esg: escargot; ISCs: intestinal stem cells; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; LLPS: liquid-liquid phase separation; LRR: leucine-rich repeat; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; p-H3: phospho-histone H3; PQ: paraquat; ref(2)P: refractory to sigma P; SQSTM1/p62: sequestosome 1; UBA: ubiquitin-associated; UPS: ubiquitin-proteasome system; VHL: von Hippel-Lindau tumor suppressor; ZER1: zyg-11 related cell cycle regulator.

The mammalian class III phosphatidylinositol-3-kinase complex (PtdIns3K) forms two biochemically and functionally distinct subcomplexes including the ATG14-containing complex I (PtdIns3K-C1) and the UVRAG-containing complex II (PtdIns3K-C2). Both subcomplexes adopt a V-shaped architecture with a BECN1-ATG14 or UVRAG adaptor arm and a PIK3R4/VPS15-PIK3C3/VPS34 catalytic arm. NRBF2 is a pro-autophagic modulator that specifically associates with PtdIns3K-C1 to enhance its kinase activity and promotes macroautophagy/autophagy. How NRBF2 exerts such a positive effect is not fully understood. Here we report that NRBF2 binds to PIK3R4/VPS15 with moderate affinity through a conserved site on its N-terminal MIT domain. The NRBF2-PIK3R4/VPS15 interaction is incompatible with the UVRAG-containing PtdIns3K-C2 because the C2 domain of UVRAG outcompetes NRBF2 for PIK3R4/VPS15 binding. Our crystal structure of the NRBF2 coiled-coil (CC) domain reveals a symmetric homodimer with multiple hydrophobic pairings at the CC interface, which is in distinct contrast to the asymmetric dimer observed in the yeast ortholog Atg38. Mutations in the CC domain that rendered NRBF2 monomeric led to weakened binding to PIK3R4/VPS15 and only partial rescue of autophagy deficiency in nrbf2 knockout cells. In comparison, NRBF2 with its CC domain replaced by a dimeric Gcn4 module showed proautophagic activity comparable to wild type while NRBF2 carrying a tetrameric Gcn4 module showed further enhanced activity. We propose that the oligomeric state of NRBF2 mediated by its CC domain is critical for strengthening the moderate NRBF2-PIK3R4/VPS15 interaction mediated by its MIT domain to fully activate PtdIns3K-C1 and promote autophagy.Abbreviations: ATG: autophagy related; ATG14: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; dCCD: delete CCD; dMIT: delete MIT; Gcn4: general control nonderepressible 4; ITC: isothermal titration calorimetry; IP: immunoprecipitation; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MIM: MIT-interacting motif; MIT: microtubule interacting and trafficking; NMR: nuclear magnetic resonance; NRBF2: nuclear receptor binding factor 2; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WT: wild type.