Autophagy最新文献

TFEB (transcription factor EB) is a critical regulator of lysosomal biogenesis, macroautophagy/autophagy and energy homeostasis through controlling expression of genes belonging to the coordinated lysosomal expression and regulation network. AMP-activated protein kinase (AMPK) has been reported to phosphorylate TFEB at three conserved C-terminal serine residues (S466, S467, S469) and these phosphorylation events were reported to be essential for transcriptional activation of TFEB. In sharp contrast to this proposition, we demonstrate that AMPK activation leads to the dephosphorylation of the C-terminal sites. We show that a synthetic peptide encompassing the C-terminal serine residues of TFEB is a poor substrate of AMPK in vitro. Treatment of cells with an AMPK activator (MK-8722), glucose deprivation or MTOR inhibitor (torin1) robustly dephosphorylated TFEB not only at the MTORC1-targeted N-terminal serine sites, but also at the C-terminal sites. Loss of function of AMPK abrogated MK-8722- but not torin1-induced dephosphorylation and induction of the TFEB target genes.Abbreviations: AMPK: 5'-adenosine monophosphate-activated protein kinase; ACAC/ACC: acetyl-CoA carboxylase; AICAR: 5-aminoimidazole-4-carbox-amide ribonucleotide; CLEAR: coordinated lysosomal expression and regulation; DKO: double knockout; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; KI: knock-in; KO: knockout; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; RRAGC: Ras related GTP binding C; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KA/RSK: ribosomal protein S6 kinase A; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Atherosclerosis is attributable to a series of diabetes-related complications. CAV1 (caveolin 1)-mediated low-density lipoprotein (LDL) particle transcytosis across endothelial cells (ECs) is the initial step of atherosclerosis. MAP1LC3/LC3-interacting regions in the intramembrane domain (IMD) of CAV1 were buried in the caveolae and were not accessible for LC3B interaction, protecting CAV1 from autophagic degradation. However, the CSD domain of CAV1, exposed in the cytosol, directly interacted with a CBM domain of LC3B and inhibited autophagy. Therefore, the peptide IMD-CBM was constructed to induce the selective autophagic degradation of CAV1 and suppress LDL transcytosis in diabetic atherosclerosis. EC-specific expression of IMD-CBM was achieved using adenovirus. IMD-CBM directly interacted with CAV1 and LC3B in ECs, leading to the selective autophagic degradation of CAV1, activation of autophagy, and subsequent inhibition of LDL transcytosis. IMD-CBM promoted the autophagic degradation of CAV1 and consequently reduced the area of atherosclerotic plaques in apoe^-/- diabetic atherosclerotic mice. Overall, IMD-CBM expedited the autophagic degradation of CAV1 and inhibited high glucose-induced LDL transcytosis, highlighting its potential as a novel translatable strategy for the management of diabetic atherosclerosis.Abbreviations: ACTB: actin beta; AKT/protein kinase B: AKT serine/threonine kinase; AMPK: 5'-adenosine monophosphate-activated protein kinase; CAV1: caveolin 1; CBM: CAV1-binding motif; CRP: C-reactive protein; CSD: CAV1-scaffolding domain; GFP: green fluorescent protein; HUVEC: human umbilical vein endothelial cell; EC: endothelial cell; FITC: fluorescein isothiocyanate; IL6: interleukin 6; IL10: interleukin 10; IMD: intramembrane domain; LDL: low-density lipoprotein; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IκBα: NFKB inhibitor alpha; NO: nitric oxide; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PIK3C3/VPS34: phosphatidylinositol-3-kinase catalytic subunit type 3; Rapa: rapamycin; SAA: serum amyloid A; SQSTM1/p62: sequestosome 1; STZ: streptozotocin; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor.

NEU (neuraminidase) is a potential cross-reactive antigen for developing broadly protective influenza vaccine, but has suboptimal immunogenicity. We here report that, when NEU antigen was redirected into phagophores, and subsequently autophagosomes, by fusing with MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; NEU-LC3B), it could efficiently activate the autophagosome-lysosome-major histocompatibility complex class II (MHC II) compartment pathway, and thus substantially improve the magnitude, breadth, and polyfunctionality of NEU-specific T cell immunity in mice. Remarkably, we identified several novel NEU-specific T-cell epitopes in response to NEU-LC3B-based immunization. Furthermore, mice immunized with NEU-based constructs were challenged with homologous A/CA/04/09 (H1N1), heterologous within-subtype strain A/Puerto Rico/8/1934 (PR8) (H1N1), and heterosubtypic A/Aichi/2/1968 (H3N2) virus, and the results demonstrated that NEU-LC3B-based vaccine provided a sterilizing immunity to homologous strains and cross-protection against antigenically distinct heterosubtypic challenge. In addition, cell depletion experiment demonstrated that T-cell-mediated immunity contributed to the NEU-LC3B-mediated immune protection. Collectively, this engineered NEU antigen with optimal immunogenicity represents a promising strategy for developing broadly protective influenza vaccines.Abbreviations: BSA, bovine serum albumin; CQ, chloroquine; ELISpot, enzyme-linked immunosorbent spot; HA, hemagglutinin; ICS, intracellular cytokine staining; IFNG/IFN-γ, interferon gamma; LD50, Median Lethal Doses; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NEU, neuraminidase; NP, nucleoprotein; PBS, phosphate-buffered saline; RAPA, rapamycin; SFC_s, spot-forming cells; SV, split vaccine; TCID₅₀, median tissue culture infectious dose; VSV, vesicular stomatitis virus; VSVΔG, VSV vector with the deletion of the G gene.

BNIP3L/NIX is a mitophagy receptor highly expressed in the brain. Unlike most mitophagy receptors that are recruited to mitochondria only upon stress, BNIP3L constitutively localizes to the mitochondrial outer membrane, suggesting functions beyond stress-induced mitophagy. Here, we identify a non-mitophagic role of BNIP3L in neuronal physiology. Conditional deletion of Bnip3l in glutamatergic neurons of the basolateral amygdala selectively impairs contextual fear memory in mice, a phenotype rescued by both wild-type BNIP3L and a mitophagy-deficient BNIP3L mutant lacking the LC3-interacting region motif. Mechanistically, BNIP3L competitively binds AMP-activated protein kinase (AMPK), thereby relieving AMPK-dependent inhibitory phosphorylation of DNM1L/DRP1 (dynamin 1 like) at Ser637. This interaction promotes rapid mitochondrial fission, supporting synaptic energy availability during memory encoding. Together, these findings reveal a switchable function of BNIP3L in neurons, acting either to acutely regulate mitochondrial dynamics to meet energetic demand or to engage mitophagy when mitochondrial function becomes compromised.

Lipophagy, the selective autophagic degradation of lipid droplets (LDs), is a key mechanism for lipid homeostasis and cellular adaptation to metabolic and stress conditions. In mammals, lipophagy is governed by signaling pathways, LD-associated receptors (e.g. SQSTM1/p62, NBR1, OPTN, SPART, OSBPL8, DDHD2, VPS4A, ATG14, and TP53INP2), and transcription factors (TFEB, TFE3, FOXO1, PPARA, PPARG, and SREBF1/SREBP1) that coordinate LD recognition, sequestration, and lysosomal degradation. Dysregulated lipophagy contributes to the pathogenesis of metabolic and age-related diseases, including metabolic dysfunction-associated steatotic liver disease/nonalcoholic fatty liver disease (MASLD/NAFLD), alcoholic liver disease, diabetes, atherosclerosis, neurodegeneration and cancer. Several recent reviews have discussed lipophagy from different angles, including its roles in metabolic disorders, central nervous system diseases, and fundamental mechanisms across species. In contrast, this review focuses specifically on mammalian lipophagy by synthesizing the latest mechanistic insights into receptor-mediated recognition, transcriptional regulation, and signaling integration. We also outline unresolved questions and conceptual gaps - such as how lipophagy is selectively activated, how it coordinates with lipolysis, and whether distinct receptor codes exist in tissue- and disease-specific contexts - that remain unanswered in the current literature.Abbreviations: AMPK, AMP-activated protein kinase; ATG, autophagy related; ATG8s: mammalian Atg8-family proteins; C1P: ceramide-1-phosphate; CMA, chaperone-mediated autophagy; COPI, coatomer protein complex I; DENV, dengue virus; ER, endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FFA: free fatty acid; HOPS, homotypic fusion and vacuole protein sorting; LDs, lipid droplets; LIR: LC3-interacting region; MASLD, metabolic dysfunction-associated steatotic liver disease; MTORC1: mechanistic target of rapamycin kinase complex 1; PE: phosphatidylethanolamine; PEDV: porcine epidemic diarrhea virus; PENV, porcine epidemic diarrhea virus; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex 1; PtdIns3P, phosphatidylinositol-3-phosphate; ROS, reactive oxygen species; SNARE: soluble NSF attachment protein receptor; SPG54: spastic paraplegia type 54; TAG: triacylglycerol/triglyceride; UBDs, ubiquitin-binding domains.

Despite the clinical success of PDCD1/PD-1 and CD274/PD-L1 immune checkpoint blockade in multiple cancers, its efficacy in colorectal cancer (CRC) remains limited. Here, we report that the combination of the tyrosine kinase inhibitor regorafenib with PDCD1 blockade enhances anti-tumor immunity in CRC, both in clinical observations and preclinical models. Mechanistically, regorafenib acts as a molecular glue, directly promoting the interaction between CD274 and the selective autophagy receptor SQSTM1/p62, leading to SQSTM1-mediated autophagic degradation of CD274 and restoration of T cell-mediated cytotoxicity. In summary, these findings identify a previously unrecognized role of regorafenib in modulating tumor immune evasion and provide a mechanistic rationale for its combination with PDCD1 inhibitors in CRC treatment.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; CD274/PD-L1: CD274 molecule; CHX: cycloheximide; co-IP: co-immunoprecipitation; CQ: chloroquine; CRC: colorectal cancer; CTLs: cytotoxic T cells; ECD: extracellular domain; GZMB: granzyme B; ICD: intracellular domain; IF: immunofluorescence; IFNG/IFN-γ: interferon gamma; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; mCRC: metastatic colorectal cancer; mIF: multiplex immunofluorescence; MSS: microsatellite stable; ORRs: objective response rates; PDCD1/PD-1: programmed cell death 1; PDCD1i: PDCD1 inhibitor; pMMR: mismatch repair-proficient; PROTACs: proteolysis-targeting chimeras; SPR: surface plasmon resonance; SQSTM1/p62: sequestosome 1; TKI: multikinase inhibitor; TME: tumor microenvironment; WB: western blot; WT: wild-type.

Alpha-herpesviruses have evolved strategies to break through immune defenses and cause severe host damage. Here, we demonstrate that the tegument protein UL48 in pseudorabies virus (PRV) inhibits type I interferon signaling by triggering STING1 degradation via a selective macroautophagy/autophagy pathway. Mechanistically, UL48 recruits the E3 ligase TRIM21 (tripartite motif containing 21), which catalyzes the ubiquitination of STING1 to form a K33/K63 linkage and is captured by the cargo receptor CALCOCO2/NDP52 for lysosomal degradation. In addition, multiple α-herpesvirus tegument protein UL48 homologs also target STING1 for degradation. Importantly, this phenotype was also observed in other herpesviruses driven by PRV UL48 homologs (herpes simplex virus-1 [HSV-1] and cercopithecine alphaherpesvirus 2 [CHV-2]). In addition, UL48-deficient PRV and HSV-1 mutant viruses attenuated pathogenicity in mice. In conclusion, this study describes a novel mechanism by which α-herpesviruses utilize UL48 proteins to promote viral escape from the host immune response.Abbreviations: 3-MA: 3-methyladenine; B-DNA: poly (dA:dT); BNIP3L/Nix: BCL2 interacting protein 3 like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; cGAMP: cyclic GMP-AMPP; CGAS: cyclic GMP-AMP synthase; CHX: cyclohexane; CHV-2: cercopithecine herpesvirus 2; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSV-1: herpes simplex virus 1; IRF3: interferon regulatory factor 3; LIR: LC3-interacting region; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MG132: cbz-leu-leu-leucinal; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PRV: pseudorabies virus; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1/STING: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TOLLIP: toll interacting protein.

Recently, mitophagy-mediated bone mineralization of mesenchymal stem cells has emerged as another bone formation pattern, but whether mitophagy-mediated bone mineralization shapes craniofacial development remains unknown. Here, we demonstrate that loss of OPTN, a keystone macroautophagy/autophagy receptor, impairs mitophagy and acidic calcium phosphate (ACP) transport in orofacial bone mesenchymal stem cells (OMSCs), leading to craniofacial bone mineralization defects. We substantiate that OPTN undergoes LLPS both in vitro and in vivo, driven by S173 phosphorylation within its intrinsically disordered N-terminal domain (NTD), facilitating the association of OPTN complexes with phagophore membranes. Additionally, the ubiquitin-binding domain (UBD) in OPTN's C-terminal domain (CTD) also promotes LLPS to recruit ubiquitin-modified mitochondria. Physiochemically, mutations at the conserved sites in human OPTN (S173A and D474N) disrupt the OPTN LLPS, as validated in mouse and zebrafish, thereby inhibiting mitophagy and impairing bone mineralization. Together, our findings reveal a new mechanism through which OPTN LLPS couples mitophagy-mediated mineralization to craniofacial bone development, highlighting its potential as a therapeutic target for treating orofacial malformations via modulation of mitophagy.Abbreviations: 1, 6HD: 1, 6-hexanediol; ACP: acidic calcium phosphate; ALP: alkaline phosphatase; ARS: Alizarin Red staining; BFR/BS: bone formation rate per bone surface; Baf-A1: bafilomycin A₁; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CTD: C-terminal domain; dpf: days post-fertilization; EDS: energy dispersive spectroscopy; FL: full length; FRAP: fluorescence recovery after photobleaching; hpf: 24h post-fertilization; IDR: intrinsically disordered region; IHC: immunohistochemistry; LLPS: liquid-liquid phase separation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MAR: mineral apposition rate; MS/BS: mineralizing surface per bone surface; NTD: N-terminal domain; ODM: osteogenic differentiation medium; OMSCs: orofacial bone mesenchymal stem cells; OPTN: optineurin; P1: postnatal day 1; P21: postnatal day 21; PDB: Paget disease of bone; PTMs: post-translational modifications; qRT-PCR: quantitative real-time PCR; S173: serine 173; STK4: serine/threonine kinase 4; SEM: scanning electron microscopy; TMD: tissue mineral density; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; Ub: ubiquitin.