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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 -
Nature Communications Mar 2023Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly...
Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.
Topics: Adolescent; Humans; Animals; Mice; Muscle, Skeletal; Autophagy; Muscular Atrophy; Macroautophagy; Myotonic Dystrophy; Mechanistic Target of Rapamycin Complex 1
PubMed: 36864049
DOI: 10.1038/s41467-023-36817-1 -
Cells Sep 2022Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely...
Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.
Topics: Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; HeLa Cells; Humans; Macroautophagy; Nuclear Proteins; Peroxisomes; Reactive Oxygen Species; Transcription Factors
PubMed: 36139416
DOI: 10.3390/cells11182839 -
The Journal of Cell Biology May 2023Macroautophagy (autophagy) utilizes a serial of receptors to specifically recognize and degrade autophagy cargoes, including damaged organelles, to maintain cellular...
Macroautophagy (autophagy) utilizes a serial of receptors to specifically recognize and degrade autophagy cargoes, including damaged organelles, to maintain cellular homeostasis. Upstream signals spatiotemporally regulate the biological functions of selective autophagy receptors through protein post-translational modifications (PTM) such as phosphorylation. However, it is unclear how acetylation directly controls autophagy receptors in selective autophagy. Here, we report that an ER-phagy receptor FAM134B is acetylated by CBP acetyltransferase, eliciting intense ER-phagy. Furthermore, FAM134B acetylation promoted CAMKII-mediated phosphorylation to sustain a mode of milder ER-phagy. Conversely, SIRT7 deacetylated FAM134B to temper its activities in ER-phagy to avoid excessive ER degradation. Together, this work provides further mechanistic insights into how ER-phagy receptor perceives environmental signals for fine-tuning of ER homeostasis and demonstrates how nucleus-derived factors are programmed to control ER stress by modulating ER-phagy.
Topics: Autophagy; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Homeostasis; Hydrolases; Macroautophagy; Membrane Proteins; Humans; Intracellular Signaling Peptides and Proteins; Sirtuins
PubMed: 37043189
DOI: 10.1083/jcb.202201068 -
Autophagy May 2020Macroautophagy (autophagy) is a key catabolic pathway for the maintenance of proteostasis through constant digestion of selective cargoes. The selectivity of autophagy...
Macroautophagy (autophagy) is a key catabolic pathway for the maintenance of proteostasis through constant digestion of selective cargoes. The selectivity of autophagy is mediated by autophagy receptors that recognize and recruit cargoes to autophagosomes. SQSTM1/p62 is a prototype autophagy receptor, which is commonly found in protein aggregates associated with major neurodegenerative diseases. While accumulation of SQSTM1 implicates a disturbance of selective autophagy pathway, the pathogenic mechanism that contributes to impaired autophagy degradation remains poorly characterized. Herein we show that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)-linked mutations of and disrupt selective autophagy and cause neurotoxicity. Our data demonstrates that proteotoxic stress activates serine/threonine kinase TBK1, which coordinates with autophagy kinase ULK1 to promote concerted phosphorylation of autophagy receptor SQSTM1 at the UBA domain and activation of selective autophagy. In contrast, ALS-FTLD-linked mutations of or reduce SQSTM1 phosphorylation and compromise ubiquitinated cargo binding and clearance. Moreover, disease mutation SQSTM1 abolishes phosphorylation of Ser351 and impairs KEAP1-SQSTM1 interaction, thus diminishing NFE2L2/Nrf2-targeted gene expression and increasing TARDBP/TDP-43 associated stress granule formation under oxidative stress. Furthermore, expression of SQSTM1 in neurons impairs dendrite morphology and KEAP1-NFE2L2 signaling. Therefore, our results reveal a mechanism whereby pathogenic SQSTM1 mutants inhibit selective autophagy and disrupt NFE2L2 anti-oxidative stress response underlying the neurotoxicity in ALS-FTLD. ALS: amyotrophic lateral sclerosis; FTLD: frontotemporal lobar degeneration; G3BP1: GTPase-activating protein (SH3 domain) binding protein 1; GSTM1: glutathione S-transferase, mu 1; HMOX/HO-1: Heme oxygenase 1; IP: immunoprecipitation; KEAP1: kelch-like ECH associated protein 1; KI: kinase inactive; KIR: KEAP1 interaction region; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; NBR1: NBR1, autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; SQSTM1/p62: sequestosome 1; SOD1: superoxide dismutase 1, soluble; S.S.: serum starvation; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; UBA: iquitin ssociation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.
Topics: Amyotrophic Lateral Sclerosis; Autophagy; Humans; Macroautophagy; Mutation; Oxidative Stress; Protein Serine-Threonine Kinases; Sequestosome-1 Protein; Signal Transduction
PubMed: 31362587
DOI: 10.1080/15548627.2019.1644076 -
The Journal of Cell Biology Oct 2022A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to...
A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).
Topics: Autophagy; Endosomes; Ferritins; Iron; Lysosomes; Nuclear Receptor Coactivators; Phagosomes; Transcription Factors
PubMed: 36066504
DOI: 10.1083/jcb.202203102 -
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