Atg9 vesicles serve as membrane seeds for autophagosome formation. These vesicles are derived from the Golgi/endosomes and localized to the pre-autophagosomal structure or phagophore assembly site (PAS) upon autophagy induction. How these vesicles are maintained as discrete membrane carriers while diffusing through the cytoplasm and subsequently become competent for downstream events at the PAS has remained unknown. Here, we show that the Atg9-interacting protein Atg23 remains associated with Atg9 vesicles following their formation and protects them from inappropriate fusion with endomembranes during their movement through the cytoplasm. Upon arrival at the PAS, Atg1-mediated phosphorylation of Atg9 triggers the dissociation of Atg23, thereby enabling efficient recruitment of the lipid-transfer protein Atg2. Collectively, these findings define a spatiotemporally regulated mechanism in which Atg23 preserves Atg9 vesicles during cytoplasmic transport, whereas its dissociation enables their productive utilization in autophagosome formation.
Sacral plexus transection (SPT), mostly caused by high‑energy trauma, induces secondary death of spinal motor neurons in the lumbosacral segments and contributes to poor clinical outcomes after nerve repair. This study aimed to investigate the protective effect of α‑ketoglutarate (AKG) against ferroptosis in spinal motor neurons following SPT and the underlying molecular mechanism. Our results showed that endogenous AKG levels were significantly decreased in the spinal cord of SPT rats and in oxidative stress‑injured motor neurons, accompanied by abnormal expression of core ferroptosis‑related proteins, including downregulated glutathione peroxidase 4 and solute carrier family 7 member 11, upregulated acyl-CoA synthetase long-chain family member 4, as well as Fe2+ overload, malondialdehyde accumulation, and glutathione depletion. Exogenous AKG supplementation markedly reversed these anomalies, suppressed ferroptosis, and improved neuronal survival. Mechanistically, AKG specifically enhanced O-GlcNAcylation at the Thr177 site of PTEN‑induced kinase 1 (PINK1) and suppressed its ubiquitin-mediated degradation, which in turn selectively activated PINK1-PRKN-dependent mitophagy to eliminate damaged mitochondria and sustain mitochondrial homeostasis. Furthermore, downregulation of isocitrate dehydrogenase 1 (IDH1) after SPT was identified as a critical upstream event leading to endogenous AKG depletion. This study systematically elucidates the key role of the IDH1‑AKG‑PINK1 O‑GlcNAcylation ‑mitophagy axis in regulating SPT‑induced ferroptosis in spinal motor neurons, providing novel targets and experimental evidence for neuroprotective therapy of sacral plexus injury.
TORC1 is a central regulator of cell growth whose inactivation under conditions of nutrient deprivation triggers adaptive responses, including macroautophagy/autophagy, amino acid uptake, and sexual differentiation. Autophagy-deficient fission yeast cells display mating defects and are unable to recover from amino acid starvation, even when external amino acids are available. Here, we investigate how TORC1 signaling and autophagy interact to control these processes. We show that both major phenotypes of autophagy-deficient cells - their inability to resume growth after amino acid starvation and their mating defects - stem from insufficient intracellular amino acid pools. Genetic or environmental enhancement of intracellular amino acid pools alleviates both defects. During leucine starvation, deletion of any1 rescues the growth defect of atg1Δ mutants by maintaining amino acid transporters at the plasma membrane, promoting amino acid uptake. Importantly, we uncover a previously unrecognized role for autophagy in the cell-cycle remodeling required for sexual differentiation. Nitrogen depletion-mediated TORC1 inactivation initiates these cell-cycle rearrangements required to start the mating/meiosis program, but autophagy is specifically required for the final G2-to-G1 arrest that precedes the program. This step correlates with the accumulation of the cyclin-dependent kinase inhibitor Rum1. Metabolomic analyses reveal that intracellular amino acid pools drop sharply during nitrogen starvation, especially in autophagy-deficient cells, and supplementation with trace amino acids restores their ability to complete the final G2-to-G1 transition. Together, our results reveal that autophagy sustains intracellular amino acid pools during prolonged stress, enabling TORC1 reactivation and cell-cycle remodeling necessary for successful mating and meiosis.Abbreviations: DNA: deoxyribonucleic acid; FACS: fluorescence-activated cell sorting; GATOR1: GAP activity toward Rags 1; GATOR2: GAP activity toward Rags 2; GFP: green fluorescent protein; MM: minimal medium; N: nitrogen; PCR: polymerase chain reaction; RNA: ribonucleic acid; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; tRNA: transfer ribonucleic acid; YE5S: yeast extract 5 amino acid supplemented; WT: wild-type.
Damaged mitochondria are selectively eliminated through mitophagy, a critical quality control process. A kinase PINK1 and an E3 ubiquitin ligase PRKN/Parkin, both of which are mutated in familial Parkinson disease, amplify ubiquitin signals on the damaged mitochondria. The autophagy receptor OPTN plays a pivotal role in mitophagy by bridging ubiquitinated mitochondria with autophagy components. Although OPTN is known to recruit ATG9A-positive vesicles to facilitate mitophagy progression, the precise molecular mechanisms governing this recruitment remain poorly understood. In this study, we identify the small RAB GTPases RAB1A and RAB1B as direct binding partners of the OPTN leucine zipper (LZ) domain. We demonstrate that RAB1A/1B is required for the recruitment of ATG9A vesicles to mitochondria during the initial stages of mitophagy. Knockdown of RAB1A and RAB1B significantly impaired the assembly of OPTN at phagophore formation sites, leading to a profound inhibition of mitophagy progression. Mechanistically, we found that RAB1A/1B associate with ATG9A-positive vesicles via their C-terminal prenylation, thereby tethering these vesicles to the OPTN-bound mitochondria. Our findings establish a novel OPTN-RAB1-ATG9A axis that drives the de novo synthesis of phagophore membranes in close proximity to damaged mitochondria. This work clarifies how selective autophagy receptors spatially coordinate membrane trafficking to ensure the efficient clearance of dysfunctional organelles.
Schizophrenia (SCZ) and bipolar disorder (BD) share genetic risk factors and cognitive impairments, yet the underlying mechanisms remain incompletely understood. Loss-of-function variants in AKAP11 (A-kinase anchoring protein 11) have recently emerged as major risk factors for both disorders. Our recent study demonstrates that AKAP11 deficiency in the mouse hippocampus causes cognitive deficits and synaptic dysfunction, accompanied by autophagy dysregulation. Mechanistically, AKAP11 interacts with PPP3CB (protein phosphatase 3 catalytic subunit beta) to promote TFEB (transcription factor EB) dephosphorylation and nuclear translocation, thereby sustaining autophagy-lysosomal gene expression. AKAP11 knockout cells display impaired autophagy initiation, reduced lysosomal activity, and compromised autophagic flux. Therapeutically, pharmacological activation of TFEB rescues cognitive deficits in Akap11-deficient mice. These findings position AKAP11 as a critical regulator of TFEB-mediated autophagy and suggest that enhancing autophagy-lysosomal function may represent a therapeutic strategy for SCZ and BD.
Thyroid hormones are central regulators of metabolic homeostasis and developmental programming. The active hormone triiodothyronine (T3) modulates transcription through nuclear receptors that recruit epigenetic cofactors to remodel chromatin and regulate metabolic gene networks. Although thyroid hormone signaling is known to influence lipid metabolism, whether it coordinates lipid droplet turnover with autophagy-related pathways during early embryonic development remains largely unknown. Here, transcriptomic profiling revealed distinct metabolic signatures between in vivo and in vitro embryos, with marked differences in fatty acid metabolism. Supplementation with 50 nM T3 enhanced blastocyst formation, particularly when applied from the 4-cell to blastocyst stages, coinciding with elevated thyroid hormone receptor expression. T3 induced robust lipid droplet remodeling, characterized by reduced droplet size, together with increased lipid-mitochondria colocalization and activation of lysosomal and mitochondrial pathways, consistent with enhanced lipid catabolism and organelle coupling. Mechanistically, inhibition of the histone acetyltransferase KAT2B/PCAF abolished T3-mediated developmental gains, reduced H3K9ac and H3K27ac, and resulted in nonselective autophagic stress rather than lipophagy. By contrast, T3 required KAT2B to stimulate cytosolic lipolysis, channel fatty acids into mitochondria, and enhance mitochondrial membrane potential. T3 also upregulated prostaglandin biosynthesis genes and improved outgrowth performance. These findings identify a thyroid hormone-KAT2B epigenetic axis that coordinates lipid droplet remodeling through lipolytic and lipophagic pathways, linking endocrine signaling to organelle crosstalk and mitochondrial activation during early embryogenesis.
Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation; however, how selective autophagy regulates ferroptotic sensitivity remains incompletely understood. Here, we identify RAB8A as a selective autophagic substrate and negative regulator of ferroptosis. Quantitative proteomic analyses reveal that ferroptotic stress induces ATG5- and ATG7-dependent degradation of RAB8A. Mechanistically, ferroptotic stimuli induce RNF126-dependent polyubiquitination of RAB8A and subsequent SQSTM1-mediated autophagic degradation. Functionally, loss of RAB8A sensitizes cancer cells to ferroptosis, whereas expression of the degradation-resistant active mutant RAB8AQ67L suppresses ferroptotic cell death. RAB8A interacts with TFRC and facilitates stress-induced redistribution of TFRC from the plasma membrane toward endolysosomal compartments. RAB8A deficiency impairs TFRC clearance, enhances transferrin-dependent iron uptake, and increases intracellular Fe2+ accumulation and lipid peroxidation. In fibrosarcoma and pancreatic cancer xenograft models, RAB8A depletion enhances the antitumor efficacy of ferroptosis-inducing therapy. Clinically, RAB8A is upregulated and associated with poor prognosis and ferroptosis resistance in pancreatic cancer. Collectively, these findings establish an autophagy-RAB8A-TFRC axis that regulates ferroptotic sensitivity.
The journal Autophagy is now in its twenty-second year. Unlike many, perhaps most, other journals we have instituted various requirements to help ensure scientific clarity and reproducibility. Two of the most important requirements are the use of standardized nomenclature and the inclusion of specific ordering information for reagents. These are not arbitrary formatting issues - there are specific reasons they are required, which we will remind you of below. The point of this editor's corner is to explain that these requirements are now going to be enforced upon manuscript submission.
Compartments of the endolysosomal and secretory pathways encounter diverse insults - from loss of ion gradients and osmotic imbalance to physical membrane disruption - creating a need for rapid, localized surveillance and response systems. Recent work demonstrates that conjugation of ATG8 to single membranes (CASM) provides such specificity via stress-responsive targeting mechanisms that recruit the ATG8 conjugation machinery through pathways distinct from macroautophagy/autophagy. Here, we review recent advances in how membrane stress is sensed, coupled to CASM initiation, and converted into downstream cellular responses.
Large-scale multiomics profiling has delineated dynamic molecular landscapes during autophagy, yet translating these complex datasets into mechanistic regulatory insights remains a major challenge. In our recent work, we developed LyMOI, a hybrid artificial intelligence workflow that combines graph-based deep learning and a large language model (LLM) for mechanistic interpretation of autophagy-related omics. The graph model integrates 1.3 TB of autophagy-associated multiomics datasets and prioritizes molecules of interest (MOIs) across 34 autophagy-specific conditions, and then LLM-based chain-of-thought (CoT) reasoning generates mechanistic hypotheses to interpret their potential roles in biological contexts. Using LyMOI, we identified essential regulators, including GIN4, ELM1, RVS167 and STE50, involved in yeast autophagy induced by nutrient deprivation. Furthermore, LyMOI revealed that two cancer-associated proteins, CTSL and FAM98A, are required for maintaining autophagy activity upon disulfiram (DSF) treatment. Silencing either CTSL or FAM98A attenuated DSF-induced autophagy and inhibited cancer cell proliferation. Notably, combination treatment with DSF and Z-FY-CHO, a CTSL-specific inhibitor previously developed against SARS-CoV-2 infection, potently suppressed tumor growth. Collectively, our work presents an LLM-powered platform with biologist-like reasoning for uncovering autophagy regulatory mechanisms.Abbreviations: ARGs: autophagy-regulating genes; ATGs: autophagy-related genes; BECN1: Beclin 1; CoT: chain-of-thought; CQ: chloroquine; CTSL: cathepsin L; DSF: disulfiram; GCNs: graph convolutional networks; GI: graph inference; IHC: immunohistochemistry; LC3-II: lipidated LC3; LGM: large graph model; LLM: large language model; MOIs: molecules of interest; NATs: normal adjacent tissues; SQSTM1/p62: sequestosome 1; 3-MA: 3-methyladenine.
BECN1 (beclin 1) is a member of the nucleation complex and considered crucial for induction of macroautophagy/autophagy, leading to the formation and ultimate degradation of autophagosomes. We found that in human B lymphoblastoid cell lines (LCLs) deficient of BECN1 (BECN1-KO), autophagosome formation was intact and autophagic flux could be induced upon nutrient starvation or MTOR inhibition. Remarkably, autophagosomal cargo differed significantly between BECN1-KO and control (CTRL) LCLs, revealing a preferred formation of autophagosomes at the endoplasmic reticulum (ER) and not at endosomes/lysosomes in BECN1-KO LCLs. Endosomal TLR3 (toll like receptor 3) was less frequently incorporated within autophagosomes in BECN1-KO LCLs. Additionally, several proteins of the ER-resident peptide loading complex for MHC class I antigen presentation were found enriched in autophagosomes from BECN1-KO LCLs, resulting in a diminished detection of BECN1-KO LCLs by T cells. Hence, BECN1 seems to be dispensable for autophagosome formation but rather contributes to cargo selection of phagophores and immunosurveillance.Abbreviations: ATG: autophagy-related; BECN1: beclin 1; CTRL: control; LCL: Epstein Barr virus transformed lymphoblastoid cell line; MHC: major histocompatibility complex; PLC: peptide-loading complex; TLR: toll like receptor.
Macroautophagy (MA) and chaperone-mediated autophagy (CMA) are lysosomal degradation pathways with context-dependent roles in cancer. However, how MA- and CMA-associated transcriptional states jointly relate to cancer molecular features and drug responses remains unclear. Here, we developed a transcriptome-based framework integrating MA- and CMA-associated gene signatures to define relative MA and CMA states across cancer types. These states were associated with distinct patterns of genomic instability, oncogenic signaling, immune features, and pharmacogenomic profiles. In pooled cancer cell-line analyses, MAhigh CMAhigh states were associated with relative resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs), whereas MAhigh CMAlow states showed greater sensitivity, nominating an autophagy-associated pharmacological pattern for mechanistic investigation. Using KRAS-mutant pancreatic cancer cells as an autophagy-dependent, therapy-resistant model, we found that LAMP2A depletion enhanced sensitivity to EGFR-TKIs in an MA-dependent manner. All-trans retinoic acid (ATRA), a pleiotropic retinoid that modulated CMA-related activity in this system, phenocopied key effects of genetic CMA suppression and potentiated sensitivity to EGFR-TKIs. CMA suppression was associated with increased autophagic flux, TFEB nuclear localization, and ULK1 phosphorylation changes consistent with MA activation. Moreover, transcriptomic analysis reveals that CMA suppression sensitizes cancer cells to EGFR-TKIs at least partially through downregulation of SEMA6D. SEMA6D depletion enhanced autophagic flux, increased lysosomal capacity, and partially contributed to the response to combined EGFR-TKI and ATRA treatment. In PANC-1 xenografts, ATRA potentiated EGFR-TKI-mediated tumor suppression without significant toxicity. Together, these findings establish a transcriptome-based MA-CMA framework for prioritizing context-dependent autophagy-associated vulnerabilities and provide focused mechanistic support for MA-CMA crosstalk in KRAS-mutant pancreatic cancer models.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ARI: adjusted rand index; ATRA: all-trans retinoic acid; BLCA: bladder urothelial carcinoma; BSA: bovine serum albumin; CMA: chaperone-mediated autophagy; CNV: copy number variation; COAD: colon adenocarcinoma; DEG: differentially expressed gene; DMEM: dulbecco's modified eagle medium; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFR: epidermal growth factor receptor; EGFR-TKI: EGFR tyrosine kinase inhibitor; EV: empty vector; FBS: fetal bovine serum; FDR: false discovery rate; FM: full medium; HCQ: hydroxychloroquine; HNSC: head and neck squamous cell carcinoma; H&E: hematoxylin and eosin; i.p.: intraperitoneally; HRD: homologous recombination deficiency; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KIRC: kidney renal clear cell carcinoma; LAMP2A: lysosome associated membrane protein 2A; LGG: lower-grade glioma; LOH: loss of heterozygosity; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MA: macroautophagy; MSI: microsatellite instability; MSigDB: molecular signatures database; OR: odds ratio; PDAC: pancreatic ductal adenocarcinoma; qRT-PCR: quantitative real-time PCR; SEMA6D: semaphorin 6D; sgRNA: single-guide RNA; siRNA: small interfering RNA; SKCM: skin cutaneous melanoma; SMD: standardized mean differences; SNV: single-nucleotide variant; SQSTM1/p62: sequestosome 1; ssGSEA: single-sample gene set enrichment analysis; STAD: stomach adenocarcinoma; TCGA: the cancer genome atlas; TMB: tumor mutation burden; TPM: transcripts per million.
Foot-and-mouth disease virus (FMDV) represents a major threat to global livestock production. The capsid protein VP1 is crucial for infection; however, the host factors and mechanisms responsible for VP1 restriction remain poorly understood. We previously identified the host chaperone DNAJA3 as a host restriction factor that inhibits FMDV infection by promoting VP1 degradation through the autophagy-lysosomal pathway. Here, we elucidate the molecular mechanism by which DNAJA3 mediates degradation of VP1. We demonstrate that DNAJA3 recruits the autophagy cargo receptor TOLLIP to facilitate selective autophagic degradation of VP1. Mechanistically, TOLLIP directly interacts with the 1-37 amino acid (aa) region of VP1 through its N-terminal and C-terminal domains, and full-length TOLLIP is required for efficient VP1 degradation. Furthermore, DNAJA3 recruits the E3 ubiquitin ligase TRIM21 to promote VP1 polyubiquitination through K27-, K48-, and K63-linked ubiquitination. TOLLIP additionally restricts FMDV internalization by modulating early endosomal trafficking in an autophagy-dependent manner. In vivo, tollip-deficient suckling mice exhibit increased susceptibility to FMDV infection. Moreover, TOLLIP exerts antiviral activity against multiple picornaviruses, including Senecavirus A and Enterovirus 71. Notably, FMDV downregulates endogenous TOLLIP expression through the protease activity of the viral 3C protein, enabling the virus to evade host autophagic surveillance. Collectively, our study identifies a novel DNAJA3-TRIM21-TOLLIP axis that restricts FMDV infection through selective autophagy, establishes TOLLIP as a host restriction factor against picornaviruses, and reveals potential molecular targets for antiviral intervention.
Endolysosomal membranes are frequently damaged by pathogenic stress associated with aging, infection, and neurodegeneration, and failure to repair such damage leads to inflammation and cell death. Recent advances identify membrane ATG8 conjugation (atg8ylation) as a key process that links damage detection to coordinated lysosomal repair, removal, and regeneration. Beyond its canonical role in macroautophagy, membrane atg8ylation also occurs on preexisting, non-autophagosomal single membranes through Conjugation of ATG8s to Single Membranes (CASM), positioning this pathway as a rapid response to membrane stress. Two E3-like ligase complexes, ATG16L1-ATG5-ATG12 and TECPR1-ATG5-ATG12, act as complementary sensors of lysosomal injury by detecting distinct physicochemical cues, including proton gradient collapse and lipid scrambling. These ligases convert damage signals into spatially restricted membrane atg8ylation, generating a membrane-associated platform that coordinates multiple downstream pathways. These include ESCRT-dependent membrane repair, ER-lysosome lipid transfer, membrane tubulation, and stress granule formation. When repair fails, membrane atg8ylation regulates lysophagy and activates lysosomal biogenesis and regeneration to restore lysosomal homeostasis. These emerging findings define membrane atg8ylation as a central organizer of membrane quality control rather than a pathway merely confined to macroautophagy. In this review, we summarize the current understanding of how membrane atg8ylation detects lysosomal damage and how this pathway coordinates other lysosomal quality control mechanisms to maintain lysosomal integrity.
Atg9-Atg2-Atg18 complexes are essential for the biogenesis of the autophagosome as they mediate the elongation of the phagophore, the precursor structure of autophagosomes. This event occurs by the transfer of lipids through a membrane contact site (MCS) between the phagophore and the endoplasmic reticulum exit sites (ERES). The bridge-like lipid-transfer protein (BLTP) Atg2 interacts with the Atg9 and phosphatidylinositol-3-phosphate (PtdIns3P) on the phagophore and acts as a tether to establish this MCS. While not essential to form the phagophore-ERES MCS, Atg18 plays a crucial role in the phagophore elongation by stimulating Atg2 lipid transfer activity, based on in vitro experiments. To understand the molecular basis of this regulation, we recently solved the structure of the yeast Atg2-Atg18 complex using cryo-electron microscopy (cryo-EM) and identified the critical region in Atg2 required for the Atg2-Atg18 complex formation. Importantly, we applied structure-function analyses to unveil the molecular mechanism behind the Atg18-mediated stimulation of Atg2. We showed that Atg18 binding to Atg2 induces a structural repositioning of the hydrophobic cavity of Atg2 toward the membrane, which allows efficient transfer of lipids from the endoplasmic reticulum to the phagophore. Here, we summarize our recent work and extend our discussion on the molecular regulation of the lipid transfer activity, highlighting open questions concerning the function of the Atg9-Atg2-Atg18 module in the phagophore-ERES MCS.Abbreviations: ATG, autophagy related; BLTP, bridge-like lipid-transfer protein; cryo-EM, cryo-electron microscopy; ER, endoplasmic reticulum; ERES, ER exit sites; MCS, membrane contact site; PAS, phagophore assembly site; PtdIns3P, phosphatidylinositol-3-phosphate; TRAPPIII, transport protein particle III.
Co-evolution between viruses and autophagy has led to the emergence of viral strategies that manipulate host endoplasmic reticulum (ER) homeostasis, ultimately promoting viral replication. ER turnover is achieved through selective autophagy, also referred to as ER-phagy, which is regulated by the RETREG1/FAM134B (reticulophagy regulator 1) family of reticulon proteins. Nevertheless, how viruses target RETREG1, a receptor for ER-phagy, remains largely unclear. In this study, we demonstrate that infection with Senecavirus A (SVA), an emerging picornavirus, triggers the cleavage of RETREG1, which functions as a negative regulator of viral replication. By screening viral proteins, we identified the SVA 3C protease (3C[pro]) as the executor of this cleavage event. Detailed mapping revealed that residues Q428, E430, and G431 of RETREG1 are involved in its cleavage by the 3C[pro], and the resulting two fragments fail to suppress viral replication. Furthermore, proteolytic cleavage of RETREG1 by 3C[pro] impairs its ability to relieve ER stress and mediate ITPR1 degradation via RETREG1-dependent ER-phagy. This disruption leads to increased ER calcium (Ca2+) release and subsequent activation of autophagy through the CAMKK2-PRKAA2-MTOR axis, which ultimately facilitates SVA replication. Taken together, these findings indicate that SVA antagonizes the antiviral function of RETREG1-mediated ER-phagy via its 3C[pro], highlighting RETREG1 as a potential therapeutic target for combating SVA infection.Abbreviations: 2-APB: 2-aminoethyl diphenylborinate; PRKAA2/AMPK: protein kinase AMP-activated catalytic subunit alpha 2; ATL3: atlastin GTPase 3; BHK-21: baby hamster kidney-21; CAMKK2: calcium/calmodulin dependent proteinkinase kinase2; CCPG1: cell cycle progression 1; CKAP4/CLIMP63: cytoskeleton associated protein 4; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DM: double mutant; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; eGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein family A (Hsp70) member 5; HA: hemagglutinin; HDAC4: histone deacetylase 4; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; IFA: indirect immunofluorescence assay; ITPR1/IP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: LC3-interacting region; mCherry: monomeric cherry; MTOR: mechanistic target of rapamycin kinase; REEP5: receptor accessory protein 5; RETREG1/FAM134B: reticulophagy regulator 1; RTN3: reticulon 3; SD: standard deviation; SEC61B: SEC61 translocon subunit beta; SEC62: SEC62 preprotein translocation factor; SERP1/RAMP4: stress associated endoplasmic reticulum protein 1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; ST: swine testis; SVA: Senecavirus A; TEM: transmission electron microscopy; TEX264: testis expressed 264, ER-phagy receptor; Tm: tunicamycin; U2OS: human osteosarcoma epithelial cells; UV: ultraviolet; ZVAD-FMK: benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; μg: microgram; μm: micrometer; μM: micromole.
Selective autophagy requires cargo receptors that not only recognize substrates but also coordinate their engagement with the autophagy machinery. Our findings identify IRGQ as a signaling-sensitive organizer of autophagy initiation rather than a passive cargo adaptor. IRGQ contains two distinct LC3-interacting region motifs: one with unusual selectivity for GABARAPL2 and another that supports broader interaction with LC3-family proteins. Proteomics, co-immunoprecipitation and imaging place the IRGQ-GABARAPL2 complex at the interface between hATG8 proteins and core autophagy-initiation components, including ATG3, ATG7, ULK1 and ATG13. Consistently, IRGQ expression promotes hATG8 lipidation and correlates with increased LC3B puncta, supporting a model in which IRGQ nucleates a local initiation hub that couples cargo recognition to autophagosome formation. Unexpectedly, this hub is negatively regulated by TBK1. TBK1-dependent phosphorylation of GABARAPL2 at serine 10 does not broadly disrupt canonical LDS-mediated interactions, but selectively destabilizes the IRGQ-GABARAPL2 complex and weakens association with autophagy-initiation factors. This phosphorylation is induced during selective-autophagy-associated conditions, including mitophagy, xenophagy and IFNγ treatment, but not during starvation-induced bulk autophagy. Functionally, GABARAPL2 S10 phosphorylation leaves LC3 and p62 bulk-autophagy readouts largely intact while reducing GABARAPL2 flux and impairing lysosomal delivery of HLA, an IRGQ cargo. Thus, TBK1 acts as a context-dependent negative regulator of a receptor-specific autophagy axis, revealing that kinase signaling can tune selective autophagy by controlling the stability and lifetime of receptor-centered initiation hubs.
SQSTM1/p62 (sequestosome 1) is an important receptor protein involved in many cellular signaling processes, including macroautophagy/autophagy. It is a molecular hub for cellular homeostasis and cellular responses. Within autophagy, SQSTM1 targets ubiquitinated cargo for degradation, maintaining cellular proteostasis. Structurally, SQSTM1 consists of several domains that facilitate its binding to ubiquitinated cargo, the formation of SQSTM1 aggregate inclusions, interactions with MAP1LC3/LC3, and the mediation of clearance via the autophagy pathway. Beyond its structure, post-translational modifications of SQSTM1 dynamically regulate its function within a cell. Post-translational modifications - such as phosphorylation, ubiquitination, acetylation, S-acylation, and S-nitrosylation - are crucial for regulating SQSTM1 function, localization, and interaction with autophagic components, thereby influencing SQSTM1's role in the autophagy pathway. Understanding the role of these protein modifications in modulating autophagy may provide better insight into developing therapeutic strategies for diseases with dysregulated autophagy, such as neurodegenerative diseases. This review will discuss the role of these post-translational modifications in controlling SQSTM1's localization and function in autophagy.Abbreviations: ABHD = α/β-hydrolase domain; AD = Alzheimer Disease; ALS = amyotrophic lateral sclerosis; ATG = autophagy related ; CSNK2/CK2 = casein kinase 2; HD = Huntington Disease; HDAC/KDAC = histone deacetylase/lysine deacetylase; HTT = huntingtin; KAT = lysine acetyltransferase; KEAP1 = kelch like ECH associated protein 1; KIR = KEAP1-interacting region; LIR = LC3-interacting region; LYPLA/APT = lysophospholipase/acyl-protein thioesterase; MAP1LC3/LC3 = microtubule associated protein 1 light chain 3; MEF = mouse embryonic fibroblast; mHTT = mutant huntingtin; MTORC1 = MTOR complex 1; NBR1 = NBR1 autophagy cargo receptor; NEDD4 = NEDD4 E3 ubiquitin protein ligase ; NO = nitric oxide; NFE2L2/Nrf2 = nuclear factor erythroid 2-factor 2; PAT = palmitoyl acyltransferase; PB1 = Phox-BEM1 domain; PE = phosphatidylethanolamine; PLEKHM1 = pleckstrin homology and RUN domain containing M1; PLK2 = polo like kinase 2; PRKA/PKA = protein kinase cAMP-activated; PPT1 = palmitoyl-protein thioesterase 1; RB1CC1 = RB1 inducible coiled-coil 1; SNCA/α-synuclein = synuclein alpha; SNO = S-nitrosothiol; SOD1 = superoxide dismutase 1; SQSTM1 = sequestosome 1; TARDBP/TDP-43 = TAR DNA binding protein ; TBK1 = TANK binding kinase 1; TAX1BP1 = Tax1 binding protein 1; TRIM = tripartite motif containing ; UBA = ubiquitin-associated domain; UBE = ubiquitin-conjugating enzyme; ULK1 = unc-51 like autophagy activating kinase 1; UPS =ubiquitin-proteasome system; USP8 = ubiquitin specific peptidase 8; ZDHHC = zDHHC palmitoyltransferase.
Selective lipophagy requires cargo recognition and recruitment of autophagy receptors to lipid droplets (LDs), yet the molecular mechanisms that couple LDs to the core autophagy machinery remain poorly defined. Here, we identified the small GTPase RAB18 (RAB18, member RAS oncogene family) as an upstream initiator of lipophagy that directly recruited the macroautophagy/autophagy receptor OPTN (optineurin) to LDs in osteoblasts. Lipid stress induced RAB18 activation and its localization to LDs, where RAB18 engaged OPTN enabling OPTN-LC3 bridging and lysosomal degradation of LDs. Loss of either RAB18 or OPTN impaired lipophagic flux, resulting in lipid accumulation and defective osteogenic differentiation, whereas OPTN overexpression partially rescued RAB18 deficiency, supporting a hierarchical RAB18-OPTN pathway. Thus, while OPTN acted as a critical effector downstream of RAB18, it did not feed back to promote RAB18 recruitment. In vivo, perturbation of this axis compromised bone regeneration under hyperlipidemic conditions. Together, these findings establish RAB18-dependent recruitment of OPTN as a molecular mechanism for selective lipophagy and reveal lipophagy as a critical metabolic adaptation that sustains osteoblast function during lipid stress.Abbreviations: AAV: adeno-associated virus; ALP: alkaline phosphatase; Baf A1: bafilomycin A1; CC domain 1: coiled-coil domain 1; CCK-8: cell counting Kit-8 kit; Co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; HE: hematoxylin and eosin; IF: immunofluorescence; IHC: immunohistochemistry; KD: knockdown; LDs: lipid droplets; Micro-CT: microscopic computerized tomography; OE: overexpression; OPTN: optineurin; qRT-PCR: quantitative real-time polymerase chain reaction; RAB18: RAB18, member RAS oncogene family; ROI: region of interest; SD: standard deviations; TBK1: TANK binding kinase 1; TEM: transmission electron microscopy; WB: western blot.
African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV), which causes severe economic losses in the swine industry. ASFV has evolved multiple strategies to evade host antiviral immune responses. Here, we report that ASFV pMGF360-3 L promotes host mitophagy by manipulating chaperone-mediated autophagy (CMA), thereby inhibiting the production of type I interferon (IFNB/IFN-β). Mechanistically, pMGF360-3 L targets the SKP1 protein via its N-terminal ankyrin (ANK) repeat domain, promoting the degradation of SKP1 through the CMA pathway, which inhibits the proteasomal degradation of BNIP3 to increase its expression level in mitochondria. Subsequently, BNIP3 binds to MAP1LC3B/LC3B to induce mitophagy, a process that leads to the degradation of mitochondria. Notably, the CMA-mediated degradation of SKP1 depends on its K94 site, and the SKP1-BNIP3 axis is critical for pMGF360-3 L-mediated IFNB inhibition. In summary, our study reveals a mechanism through which ASFV pMGF360-3 L facilities CMA-dependent degradation of the E3 complex component SKP1. This stabilizes mitochondrial BNIP3 to initiate mitophagy and block IFNB production. This deepens our understanding of the immune evasion strategies of ASFV and provides potential drug targets for controlling viral infection.Abbreviations: 3-MA: 3-methyladenine; ASFV: African swine fever virus; BafA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CQ: chloroquine; CHX: cycloheximide; CUL1: cullin 1; DAPI: 4', 6-diamidino-2'-phenylindole; EV: emptor vector; FBXL4: F-box and leucine rich repeat protein 4; hpi: hours post-infection; IFNB: interferon beta; ISGs: IFN-stimulated genes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MG132: cbz-leu-leu-leucinal; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMs: porcine alveolar macrophages; PPTC7: protein phosphatase targeting COQ7; RBX1: ring-box 1; RT-PCR: real-time polymerase chain reaction; siRNA: small interfering RNA; SKP1: S-phase kinase associated protein 1; TCID50: 50% tissue culture infectious doses; Ub: ubiquitin; WCL: whole-cell lysate; WT: wild-type.
