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Autophagy Aug 2021TMEM41B and VMP1, two endoplasmic reticulum (ER)-resident transmembrane proteins, play important roles in regulating the formation of lipid droplets (LDs), autophagy... (Review)
Review
TMEM41B and VMP1, two endoplasmic reticulum (ER)-resident transmembrane proteins, play important roles in regulating the formation of lipid droplets (LDs), autophagy initiation, and viral infection. However, the biochemical functions of TMEM41B and VMP1 are unclear. A lipids distribution screen suggested TMEM41B and VMP1 are critical to the normal distribution of cholesterol and phosphatidylserine. Biochemical analyses unveiled that TMEM41B and VMP1 have scramblase activity. These findings shed light on the mechanism by which TMEM41B and VMP1 regulate LD formation, lipids distribution, macroautophagy, and viral infection.
Topics: Animals; Autophagosomes; Autophagy; Humans; Macroautophagy; Membrane Proteins; Phospholipid Transfer Proteins
PubMed: 34074213
DOI: 10.1080/15548627.2021.1937898 -
Neuron Aug 2023Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and...
Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and behavioral deficits are largely recapitulated in mouse models with ablation of autophagy genes in brain cells. Yet, the nature and temporal dynamics of brain autophagic substrates remain insufficiently characterized. Here, we immunopurified LC3-positive autophagic vesicles (LC3-pAVs) from the mouse brain and proteomically profiled their content. Moreover, we characterized the LC3-pAV content that accumulates after macroautophagy impairment, validating a brain autophagic degradome. We reveal selective pathways for aggrephagy, mitophagy, and ER-phagy via selective autophagy receptors, and the turnover of numerous synaptic substrates, under basal conditions. To gain insight into the temporal dynamics of autophagic protein turnover, we quantitatively compared adolescent, adult, and aged brains, revealing critical periods of enhanced mitophagy or degradation of synaptic substrates. Overall, this resource unbiasedly characterizes the contribution of autophagy to proteostasis in the maturing, adult, and aged brain.
Topics: Animals; Mice; Autophagy; Mitophagy; Macroautophagy; Aging; Brain
PubMed: 37279748
DOI: 10.1016/j.neuron.2023.05.011 -
The Journal of Cell Biology Dec 2023Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by...
Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.
Topics: Autophagy; Lysosomes; Macroautophagy; Microtubule-Associated Proteins; Salmonella; Autophagy-Related Protein 8 Family
PubMed: 37796195
DOI: 10.1083/jcb.202303078 -
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 -
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 -
Current Biology : CB Jul 2019In 1955, the biologist and Nobel Prize laureate Christian de Duve discovered that cells possess specialized organelles filled with hydrolytic enzymes and he called these...
In 1955, the biologist and Nobel Prize laureate Christian de Duve discovered that cells possess specialized organelles filled with hydrolytic enzymes and he called these organelles lysosomes. At the same time, electron microscopy studies by Novikoff and colleagues showed that intracellular dense bodies, which later turned out to be lysosomes, contain cytoplasmic components. Together, these groundbreaking observations revealed that cells can deliver cytoplasmic components to lysosomes for degradation. The hallmark of this degradative process, which de Duve called autophagy, is the formation of double-membrane-limited vesicles. Further morphological characterization of these vesicles (autophagosomes) revealed that they mainly contain bulk cytoplasm. Although this suggested that autophagy leads to a non-selective degradation of cytoplasmic material, de Duve anticipated that a regulated and selective type of this pathway must also exist. Today we know that, under normal conditions, macroautophagy is a highly selective pathway that sequesters damaged or superfluous material from the cytoplasm through the formation of double-membrane-limited autophagosomes. Upon fusion with lysosomes, the content of autophagosomes is degraded and the resulting building blocks are released into the cytoplasm. However, in response to cytotoxic stress or starvation, cells start to produce autophagosomes that capture bulk cytoplasm non-selectively. This stress response is essential for cells to survive adverse environmental conditions, whereas the selective sequestration of cargo is important to maintain cellular homeostasis.
Topics: Autophagosomes; Autophagy; Cytosol; Lysosomes; Macroautophagy
PubMed: 31336079
DOI: 10.1016/j.cub.2019.06.014 -
Cell Death and Differentiation Jan 2020Lipophagy is a lysosomal lipolytic pathway that complements the actions of cytosolic neutral lipases. Chaperon-mediated autophagy (CMA) triggers lipid droplets (LDs)...
Lipophagy is a lysosomal lipolytic pathway that complements the actions of cytosolic neutral lipases. Chaperon-mediated autophagy (CMA) triggers lipid droplets (LDs) breakdown, to initiate lipolysis via either cytosolic lipases or macroautophagy. SIRT3, a mitochondrial NAD-dependent deacetylase, regulates the acetylation status and activity of many substrates involving in energy metabolism. However, the role of SIRT3 in regulating lipophagy is controversial. The current study showed that SIRT3 expression was decreased and the macroautophagy flux was blocked in the primary hepatocytes from high-fat diet fed mice and P/O (palmitic acid and oleic acid mixture) treated AML12 mouse hepatocytes, compared with the corresponding controls. SIRT3 overexpression promoted macroautophagy in LDs from P/O-treated hepatocytes through activating AMP-activated protein kinase (AMPK) and unc-51-like kinase 1, to boost LDs digestion. Gain of SIRT3 expression stimulated the formation of lysosome-associated membrane protein 2A (LAMP-2A)-heat shock cognate 71 kDa protein (HSC70)-perilipin-2 (PLN2) complex, to promote CMA process and reduce the stability of LDs in hepatocytes. Moreover, SIRT3 reduced the expression of stearoyl-CoA desaturase 1, to suppress lipogenesis. In addition, SIRT3 overexpression promoted LDs dispersion on detyrosinated microtubules, and directly deacetylated long-chain acyl-CoA dehydrogenase to enhance mitochondrial energetics. Taken together, SIRT3 ameliorates lipotoxicity in hepatocytes, which might be a potential target for the treatment of nonalcoholic fatty liver disease.
Topics: AMP-Activated Protein Kinase Kinases; Animals; Autophagy-Related Protein-1 Homolog; Cell Line; Chaperone-Mediated Autophagy; Diet, High-Fat; Hepatocytes; Lipid Droplets; Lipid Metabolism; Lipolysis; Macroautophagy; Male; Mice, Inbred C57BL; Microtubules; Mitochondria; Protein Kinases; Sirtuin 3; Stearoyl-CoA Desaturase
PubMed: 31160717
DOI: 10.1038/s41418-019-0356-z -
Autophagy Jun 2022Mitophagy is a selective autophagy mechanism for eliminating damaged mitochondria and plays a crucial role in the immune evasion of some viruses and bacteria. Here, we...
Mitophagy is a selective autophagy mechanism for eliminating damaged mitochondria and plays a crucial role in the immune evasion of some viruses and bacteria. Here, we report that () utilizes host mitophagy to suppress host xenophagy to enhance its intracellular survival. is the causative agent of animal tuberculosis and human tuberculosis. In the current study, we show that induces mitophagy in macrophages, and the induction of mitophagy is impaired by PINK1 knockdown, indicating the PINK1-PRKN/Parkin pathway is involved in the mitophagy induced by . Moreover, the survival of in macrophages and the lung bacterial burden of mice are restricted by the inhibition of mitophagy and are enhanced by the induction of mitophagy. Confocal microscopy analysis reveals that induction of mitophagy suppresses host xenophagy by competitive utilization of p-TBK1. Overall, our results suggest that induction of mitophagy enhances growth while inhibition of mitophagy improves growth restriction. The findings provide a new insight for understanding the intracellular survival mechanism of in the host. BMDM: mouse bone marrow-derived macrophage; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; BCL2L13: BCL2-like 13 (apoptosis facilitator); CCCP: carbonyl cyanide m-cholorophenyl hydrazone; FUNDC1: FUN14 domain-containing 1; FKBP8: FKBP506 binding protein 8; HCV: hepatitis C virus; HBV: hepatitis B virus; IFN: interferon; ; Mtb: ; Mdivi-1: mitochondrial division inhibitor 1; PINK1: PTEN-induced putative kinase 1; TBK1: TANK-binding kinase 1; TUFM: Tu translation elongation factor, mitochondrial; TEM: transmission electron microscopy.
Topics: Animals; Macroautophagy; Macrophages; Membrane Proteins; Mice; Mitochondrial Proteins; Mitophagy; Mycobacterium bovis
PubMed: 34720021
DOI: 10.1080/15548627.2021.1987671 -
Autophagy Dec 2021Macroautophagy/autophagy, an evolutionarily conserved process, plays an important role in the regulation of immune inflammation and nervous system homeostasis. However,...
Macroautophagy/autophagy, an evolutionarily conserved process, plays an important role in the regulation of immune inflammation and nervous system homeostasis. However, the exact role and mechanism of autophagy in pain is still unclear. Here, we showed that impaired autophagy flux mainly occurred in astrocytes during the maintenance of neuropathic pain. No matter the stage of neuropathic pain induction or maintenance, activation of autophagy relieved the level of pain, whereas inhibition of autophagy aggravated pain. Moreover, the levels of neuroinflammation and reactive oxygen species (ROS) were increased or decreased following autophagy inhibition or activation. Further study showed that inhibition of autophagy slowed the induction, but increased the maintenance of neuroinflammatory responses, which could be achieved by promoting the binding of TRAF6 (TNF receptor-associated factor 6) to K63 ubiquitinated protein, and increasing the levels of p-MAPK8/JNK (mitogen-activated protein kinase 8) and nuclear factor of kappa light polypeptide gene enhancer in B cells (NFKB/NF-κB). Impaired autophagy also reduced the protective effect of astrocytes on neurons against ROS stress because of the decrease in the level of glutathione released by astrocytes, which could be improved by activating the NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) pathway. We also demonstrated that simultaneous activation of autophagy and the NFE2L2 pathway further relieved pain, compared to activating autophagy alone. Our study provides an underlying mechanism by which autophagy participates in the regulation of neuropathic pain, and a combination of autophagy and NFE2L2 activation may be a new treatment approach for neuropathic pain. 3-MA: 3-methyladenine; 8-OHdG: 8-hydroxydeoxy-guanosine; ACTB: actin, beta; AMPAR: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor; ATG: autophagy-related; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CCL7: chemokine (C-C motif) ligand 7; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; GABA: gamma-aminobutyrate; GCLC: glutamate-cysteine ligase, catalytic subunit; GFAP: glial fibrillary acidic protein; GSH: glutathione; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch-like ECH-associated protein 1; MAP1LC3/LC3-II: microtubule-associated protein 1 light chain 3 beta (phosphatidylethanolamine-conjugated form); MAPK: mitogen-activated protein kinase; MAPK1/ERK: mitogen-activated protein kinase 1; MMP2: matrix metallopeptidase 2; MAPK8/JNK: mitogen-activated protein kinase 8; MAPK14/p38: mitogen-activated protein kinase 14; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; ROS: reactive oxygen species; SLC12A5: solute carrier family 12, member 5; SNL: spinal nerve ligation; TLR4: toll-like receptor 4; TRAF6: TNF receptor-associated factor; TRP: transient receptor potential.
Topics: Autophagy; Humans; Kelch-Like ECH-Associated Protein 1; Macroautophagy; NF-E2-Related Factor 2; Neuralgia
PubMed: 33834930
DOI: 10.1080/15548627.2021.1900498 -
Autophagy Aug 2022Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical...
Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical roles in immunity and other cellular functions. To investigate the role of USPs in alphaherpesvirus replication, we assessed 13 USP inhibitors for PRV replication. Our data showed that all the tested compounds inhibited PRV replication, with the USP14 inhibitor b-AP15 exhibiting the most dramatic effect. Ablation of USP14 also influenced PRV replication, whereas replenishment of USP14 in null cells restored viral replication. Although inhibition of USP14 induced the K63-linked ubiquitination of PRV VP16 protein, its degradation was not dependent on the proteasome. USP14 directly bound to ubiquitin chains on VP16 through its UBL domain during the early stage of viral infection. Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy. Ectopic expression of non-ubiquitinated VP16 fully rescued PRV replication. Challenge of mice with b-AP15 activated ER stress and autophagy and inhibited PRV infection . Our results suggested that USP14 was a potential therapeutic target to treat alphaherpesvirus-induced infectious diseases. ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; CCK-8: cell counting kit-8; Co-IP: co-immunoprecipitation; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR associated system 9; DDIT3/CHOP: DNA-damage inducible transcript 3; DNAJB9/ERdj4: DnaJ heat shock protein family (Hsp40) member B9; DUBs: deubiquitinases; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EP0: ubiquitin E3 ligase ICP0; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; FOXO1: forkhead box O1; FRET: Förster resonance energy transfer; HSPA5/BiP: heat shock protein 5; HSV: herpes simplex virus; IE180: transcriptional regulator ICP4; MAP1LC3/LC3: microtube-associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; PRV: pseudorabies virus; PRV gB: PRV glycoprotein B; PRV gE: PRV glycoprotein E; qRT-PCR: quantitative real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID: tissue culture infective dose; UB: ubiquitin; UBA: ubiquitin-associated domain; UBL: ubiquitin-like domain; UL9: DNA replication origin-binding helicase; UPR: unfolded protein response; USPs: ubiquitin-specific proteases; VHS: virion host shutoff; VP16: viral protein 16; XBP1: X-box binding protein 1; XBP1s: small XBP1; XBP1(t): XBP1-total.
Topics: Alphaherpesvirinae; Animals; Autophagy; Cell Proliferation; Endoplasmic Reticulum Stress; Herpes Simplex Virus Protein Vmw65; Macroautophagy; Mice; Sequestosome-1 Protein; Ubiquitin Thiolesterase
PubMed: 34822318
DOI: 10.1080/15548627.2021.2002101