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Cell Jan 2017The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor...
The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.
Topics: Aging; Animals; Autophagosomes; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Embryo, Nonmammalian; Membrane Proteins; Mitochondrial Membranes; Prohibitins; Repressor Proteins
PubMed: 28017329
DOI: 10.1016/j.cell.2016.11.042 -
Cell Research Dec 2022STING, an endoplasmic reticulum (ER) transmembrane protein, mediates innate immune activation upon cGAMP stimulation and is degraded through autophagy. Here, we report...
STING, an endoplasmic reticulum (ER) transmembrane protein, mediates innate immune activation upon cGAMP stimulation and is degraded through autophagy. Here, we report that activated STING could be transferred between cells to promote antitumor immunity, a process triggered by RAB22A-mediated non-canonical autophagy. Mechanistically, RAB22A engages PI4K2A to generate PI4P that recruits the Atg12-Atg5-Atg16L1 complex, inducing the formation of ER-derived RAB22A-mediated non-canonical autophagosome, in which STING activated by agonists or chemoradiotherapy is packaged. This RAB22A-induced autophagosome fuses with RAB22A-positive early endosome, generating a new organelle that we name Rafeesome (RAB22A-mediated non-canonical autophagosome fused with early endosome). Meanwhile, RAB22A inactivates RAB7 to suppress the fusion of Rafeesome with lysosome, thereby enabling the secretion of the inner vesicle of the autophagosome bearing activated STING as a new type of extracellular vesicle that we define as R-EV (RAB22A-induced extracellular vesicle). Activated STING-containing R-EVs induce IFNβ release from recipient cells to the tumor microenvironment, promoting antitumor immunity. Consistently, RAB22A enhances the antitumor effect of the STING agonist diABZI in mice, and a high RAB22A level predicts good survival in nasopharyngeal cancer patients treated with chemoradiotherapy. Our findings reveal that Rafeesome regulates the intercellular transfer of activated STING to trigger and spread antitumor immunity, and that the inner vesicle of non-canonical autophagosome originated from ER is secreted as R-EV, providing a new perspective for understanding the intercellular communication of organelle membrane proteins.
Topics: Animals; Mice; Autophagosomes; Autophagy; Immunity, Innate; Lysosomes; Membrane Proteins; Nasopharyngeal Neoplasms; Tumor Microenvironment; Humans
PubMed: 36280710
DOI: 10.1038/s41422-022-00731-w -
Journal of Molecular Biology Jan 2020Selective autophagy relies on soluble or membrane-bound cargo receptors that recognize cargo and bring about autophagosome formation at the cargo. The cargo-bound... (Review)
Review
Selective autophagy relies on soluble or membrane-bound cargo receptors that recognize cargo and bring about autophagosome formation at the cargo. The cargo-bound receptors interact with lipidated ATG8 family proteins anchored in the membrane at the concave side of the forming autophagosome. The interaction is mediated by 15- to 20-amino-acid-long sequence motifs called LC3-interacting region (LIR) motifs that bind to the LIR docking site (LDS) of ATG8 proteins. In this review, we focus on LIR-ATG8 interactions and the soluble mammalian selective autophagy receptors. We discuss the roles of ATG8 family proteins as membrane scaffolds in autophagy and the LIR-LDS interaction and how specificity for binding to GABARAP or LC3 subfamily proteins is achieved. We also discuss atypical LIR-LDS interactions and a novel LIR-independent interaction. Recently, it has become clear that several of the soluble cargo receptors are able to recruit components of the core autophagy apparatus to aid in assembling autophagosome formation at the site of cargo sequestration. A model on phagophore recruitment and expansion on a selective autophagy receptor-coated cargo incorporating the latest findings is presented.
Topics: Animals; Apoptosis Regulatory Proteins; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Humans; Macroautophagy; Microtubule-Associated Proteins; Protein Interaction Domains and Motifs; Protein Interaction Maps
PubMed: 31310766
DOI: 10.1016/j.jmb.2019.07.016 -
The EMBO Journal Apr 2023The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism...
The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation.
Topics: Autophagosomes; Autophagy; DNA; Membrane Proteins; Nucleotidyltransferases; Humans
PubMed: 36872914
DOI: 10.15252/embj.2022112387 -
Autophagy Jan 2023SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of...
SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of acetylation (a post-translational modification) on macroautophagy/autophagy. Here, we demonstrate that both acetylation- and phosphorylation-dependent mechanisms control SCFD1's function in autophagosome-lysosome fusion. After detecting a decrease in the extent of SCFD1 acetylation under autophagy-stimulated conditions, we found that KAT2B/PCAF catalyzes the acetylation of residues K126 and K515 of SCFD1; we also showed that these two residues are deacetylated by SIRT4. Importantly, we showed that AMPK-controlled SCFD1 phosphorylation strongly disrupts the capacity of SCFD1 to interact with KAT2B, thus ensuring that the SCFD1 acetylation level remains low. Finally, we demonstrated that SCFD1 acetylation inhibits autophagic flux, specifically by blocking STX17-SNAP29-VAMP8 SNARE complex formation. Thus, our study reveals a mechanism through which phosphorylation and acetylation modifications of SCFD1 mediate SNARE complex formation to regulate autophagosome maturation.ACLY: ATP citrate lyase; CREB: cAMP responsive element binding protein; EBSS: nutrient-deprivation medium; EP300: E1A binding protein p300; KAT5/TIP60: lysine acetyltransferase 5; HOPS: homotypic fusion and protein sorting; MS: mass spectroscopy; SCFD1: sec1 family domain containing 1; SM: Sec1/Munc18; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UVRAG: UV radiation resistance associated.
Topics: Autophagy; Autophagosomes; Macroautophagy; Acetylation; Lysosomes; Protein Processing, Post-Translational; SNARE Proteins; Membrane Fusion
PubMed: 35465820
DOI: 10.1080/15548627.2022.2064624 -
The Journal of Cell Biology Jun 2022The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs)....
The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.
Topics: Autophagosomes; Autophagy; Extracellular Vesicles; Humans; Lysosomes; Proteostasis; SARS-CoV-2; Sequestosome-1 Protein; Viroporin Proteins; rab27 GTP-Binding Proteins
PubMed: 35446347
DOI: 10.1083/jcb.202110151 -
Cell Research Sep 2021Degrading pathogenic proteins by degrader technologies such as PROTACs (proteolysis-targeting chimeras) provides promising therapeutic strategies, but selective...
Degrading pathogenic proteins by degrader technologies such as PROTACs (proteolysis-targeting chimeras) provides promising therapeutic strategies, but selective degradation of non-protein pathogenic biomolecules has been challenging. Here, we demonstrate a novel strategy to degrade non-protein biomolecules by autophagy-tethering compounds (ATTECs), using lipid droplets (LDs) as an exemplar target. LDs are ubiquitous cellular structures storing lipids and could be degraded by autophagy. We hypothesized that compounds interacting with both the LDs and the key autophagosome protein LC3 may enhance autophagic degradation of LDs. We designed and synthesized such compounds by connecting LC3-binding molecules to LD-binding probes via a linker. These compounds were capable of clearing LDs almost completely and rescued LD-related phenotypes in cells and in two independent mouse models with hepatic lipidosis. We further confirmed that the mechanism of action of these compounds was mediated through LC3 and autophagic degradation. Our proof-of-concept study demonstrates the capability of degrading LDs by ATTECs. Conceptually, this strategy could be applied to other protein and non-protein targets.
Topics: Animals; Autophagosomes; Autophagy; Chimera; Lipid Droplets; Lipid Metabolism; Mice; Proteins
PubMed: 34239073
DOI: 10.1038/s41422-021-00532-7 -
Cell Oct 2022The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca transients on the...
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca transients. Multi-modal SIM analysis revealed that Ca transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Topics: Animals; Autophagosomes; Calcium; Endoplasmic Reticulum; Autophagy-Related Proteins; Autophagy; Cell Cycle Proteins
PubMed: 36198318
DOI: 10.1016/j.cell.2022.09.001 -
The Journal of Cell Biology Aug 2023Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also...
Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.
Topics: Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Macroautophagy; Phosphorylation; Humans; Qa-SNARE Proteins; Filamins
PubMed: 37389864
DOI: 10.1083/jcb.202211025 -
The Journal of Cell Biology Jul 2023As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the...
As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.
Topics: Animals; Autophagosomes; Membrane Proteins; Autophagy; Protein Transport; Autophagy-Related Proteins; Lipids; Mammals
PubMed: 37115958
DOI: 10.1083/jcb.202208088