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Protein & Cell Sep 2023Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that...
Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on LDs and is phosphorylated by AMPK, thereby enhancing its affinity for LC3/GABARAPs. Deletion of ORP8 or interruption of ORP8-LC3/GABARAP interaction results in accumulation of LDs and increased intracellular triglyceride. Overexpression of ORP8 alleviates LD and triglyceride deposition in the liver of ob/ob mice, and Osbpl8-/- mice exhibit liver lipid clearance defects. Our results suggest that ORP8 is a lipophagy receptor that plays a key role in cellular lipid metabolism.
Topics: Animals; Mice; Lipid Droplets; Autophagy; Autophagosomes; Homeostasis; Triglycerides
PubMed: 37707322
DOI: 10.1093/procel/pwac063 -
Cell Feb 2024Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat...
Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.
Topics: Animals; Female; Mice; Autophagosomes; Cytoplasmic Vesicles; Lysosomes; Oocytes; Proteasome Endopeptidase Complex; Protein Aggregates; Proteolysis
PubMed: 38382525
DOI: 10.1016/j.cell.2024.01.031 -
Cell Apr 2024Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional...
Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.
Topics: Animals; Mice; Interferon Type I; Microglia; Neurons; Zebrafish; Brain
PubMed: 38490196
DOI: 10.1016/j.cell.2024.02.020 -
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 -
Autophagy Jul 2023Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer...
Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.
Topics: Autophagosomes; Autophagy; Macroautophagy; Reactive Oxygen Species; Lysosomes; SNARE Proteins; Apoptosis
PubMed: 36704963
DOI: 10.1080/15548627.2023.2170962 -
The Journal of Cell Biology Sep 2023Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key...
Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.
Topics: Animals; Dictyostelium; Endosomes; Mammals; Phagosomes; Phosphatidylinositols; Phosphatidylinositol 3-Kinases
PubMed: 37382666
DOI: 10.1083/jcb.202209077 -
Molecular Cell Oct 2023p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal...
p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.
Topics: Mice; Rats; Humans; Animals; Autophagosomes; Ubiquitinated Proteins; Sequestosome-1 Protein; Autophagy; Acylation; Microtubule-Associated Proteins; Mammals
PubMed: 37802024
DOI: 10.1016/j.molcel.2023.09.004 -
International Journal of Molecular... Aug 2023Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt...
Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt reabsorption and thus explaining the transient variant of Bartter's syndrome. The cAMP/PKA pathway is also known to regulate autophagy, a lysosomal degradation process induced by cellular stress. Previous studies showed that two members of the melanoma-associated antigens MAGE-family inhibit autophagy. To explore the potential role of MAGED2 in stress-induced autophagy, specific MAGED2-siRNA were used in HEK293 cells under physical hypoxia and oxidative stress (cobalt chloride, hypoxia mimetic). Depletion of MAGED2 resulted in reduced p62 levels and upregulation of both the autophagy-related genes (ATG5 and ATG12) as well as the autophagosome marker LC3II compared to control siRNA. The increase in the autophagy markers in MAGED2-depleted cells was further confirmed by leupeptin-based assay which concurred with the highest LC3II accumulation. Likewise, under hypoxia, immunofluorescence in HEK293, HeLa and U2OS cell lines demonstrated a pronounced accumulation of LC3B puncta upon MAGED2 depletion. Moreover, LC3B puncta were absent in human fetal control kidneys but markedly expressed in a fetal kidney from a MAGED2-deficient subject. Induction of autophagy with both physical hypoxia and oxidative stress suggests a potentially general role of MAGED2 under stress conditions. Various other cellular stressors (brefeldin A, tunicamycin, 2-deoxy-D-glucose, and camptothecin) were analyzed, which all induced autophagy in the absence of MAGED2. Forskolin (FSK) inhibited, whereas GNAS Knockdown induced autophagy under hypoxia. In contrast to other MAGE proteins, MAGED2 has an inhibitory role on autophagy only under stress conditions. Hence, a prominent role of MAGED2 in the regulation of autophagy under stress conditions is evident, which may also contribute to impaired fetal renal salt reabsorption by promoting autophagy of salt-transporters in patients with MAGED2 mutation.
Topics: Humans; HEK293 Cells; Autophagy; Oxidative Stress; Autophagosomes; Sodium Chloride; Sodium Chloride, Dietary; Melanoma; Antigens, Neoplasm; Adaptor Proteins, Signal Transducing
PubMed: 37686237
DOI: 10.3390/ijms241713433 -
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 -
Autophagy Jan 2024Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is...
Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is not known. Omegasomes are characterized by the presence of the double FYVE domain containing protein ZFYVE1/DFCP1, but it has remained a paradox that depletion of ZFYVE1 does not prevent bulk macroautophagy/autophagy. We recently showed that ZFYVE1 contains an N-terminal ATPase domain which dimerizes upon ATP binding. Mutations in the ATPase domain that inhibit ATP binding or hydrolysis do not prevent omegasome expansion and maturation. However, omegasome constriction is inhibited by these mutations, which results in an increased lifetime and thereby higher number of omegasomes. Interestingly, whereas knockout or mutations do not significantly affect bulk autophagy, selective autophagy of mitochondria, protein aggregates and micronuclei is inhibited. We propose that ATP binding and hydrolysis control the di- or multimerization state of ZFYVE1 which could provide the mechanochemical energy to drive large omegasome constriction and autophagosome completion.
Topics: Autophagy; Autophagosomes; Macroautophagy; Adenosine Triphosphatases; Adenosine Triphosphate
PubMed: 37722386
DOI: 10.1080/15548627.2023.2255967