Autophagy最新文献

The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A₁; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole.

The DNA damage response (DDR) pathway is a cardinal cellular stress response mechanism that during cancer development follows an antagonistic pleiotropy mode of action. Given that DDR activation is an energy demanding process, interplay with macroautophagy/autophagy, a stress response and energy providing mechanism, is likely to take place. While molecular connections between both mechanisms have been reported, an open question regards whether autophagy activation follows solely or is entangled with DDR in a similar antagonistic pleiotropy pattern during cancer development. Combing evidence on the spatiotemporal relationship of DDR and autophagy in the entire spectrum of carcinogenesis from our previous studies, we discuss these issues in the current addendum.Abbreviation: AMPK: AMP-dependent protein kinase; DDR: DNA damage response.

Macroautophagic/autophagic and endocytic pathways play essential roles in maintaining homeostasis at different levels. It remains poorly understood how both pathways are coordinated and fine-tuned for proper lysosomal degradation of diverse cargoes. We and others recently identified a Golgi-resident RAB GTPase, RAB2A, as a positive regulator that controls both autophagic and endocytic pathways. In the current study, we report that TBC1D4 (TBC1 domain family member 4), a TBC domain-containing protein that plays essential roles in glucose homeostasis, suppresses RAB2A-mediated autophagic and endocytic pathways. TBC1D4 bound to RAB2A through its N-terminal PTB2 domain, which impaired RAB2A-mediated autophagy at the early stage by preventing ULK1 complex activation. During the late stage of autophagy, TBC1D4 impeded the association of RUBCNL/PACER and RAB2A with STX17 on autophagosomes by direct interaction with RUBCNL via its N-terminal PTB1 domain. Disruption of the autophagosomal trimeric complex containing RAB2A, RUBCNL and STX17 resulted in defective HOPS recruitment and eventually abortive autophagosome-lysosome fusion. Furthermore, TBC1D4 inhibited RAB2A-mediated endocytic degradation independent of RUBCNL. Therefore, TBC1D4 and RAB2A form a dual molecular switch to modulate autophagic and endocytic pathways. Importantly, hepatocyte- or adipocyte-specific tbc1d4 knockout in mice led to elevated autophagic flux and endocytic degradation and tissue damage. Together, this work establishes TBC1D4 as a critical molecular brake in autophagic and endocytic pathways, providing further mechanistic insights into how these pathways are intertwined both in vitro and in vivo.Abbreviations: ACTB: actin beta; ATG9: autophagy related 9; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; CLEM: correlative light electron microscopy; Ctrl: control; DMSO: dimethyl sulfoxide; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FL: full length; GAP: GTPase-activating protein; GFP: green fluorescent protein; HOPS: homotypic fusion and protein sorting; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OE: overexpression; PG: phagophore; PtdIns3K: class III phosphatidylinositol 3-kinase; SLC2A4/GLUT4: solute carrier family 2 member 4; SQSTM1/p62: sequestosome 1; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TAP: tandem affinity purification; TBA: total bile acid; TBC1D4: TBC1 domain family member 4; TUBA1B: tubulin alpha 1b; ULK1: unc-51 like autophagy activating kinase 1; VPS39: VPS39 subunit of HOPS complex; WB: western blot; WT: wild type.

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.Abbreviations: Baf-A1, bafilomycin A₁; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DBCO, dibenzocyclooctyne; ECAM, enzyme-activated probe covalently attached on mitochondrial inner membrane; GFP, green fluorescent protein; LAMP2, lysosomal associated membrane protein 2; LNPEP/LAP, leucyl and cystinyl aminopeptidase; PMA, phorbol-12-myristate-13-acetate; ΔΨm, mitochondrial transmembrane potential; RFP, red fluorescent protein; TPP, triphenylphosphonium.

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A₁; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium.

In eukaryotic cells, membrane contact sites (MCSs) mediate interactions and communication between organelles by bringing their membranes into close proximity without fusion. These sites play crucial roles in intracellular transport, signal transduction, and the regulation of organelle functions. In a recent study, we compiled data on MCS proteins and complexes from publications to create the MCSdb database. During data compilation, we discovered that many MCSs, their associated proteins, and complexes are highly relevant to macroautophagy/autophagy. To elucidate the role of MCSs in autophagy, we reorganized the autophagy-related MCS proteins and complexes from MCSdb, creating a data map called AutoMCS Navigator. The current version of this map includes 30 complexes and 84 proteins, covering 13 different MCSs and 7 species. Meanwhile, we embedded a dedicated webpage for AutoMCS Navigator on the MCSdb website. This webpage features an orchestrated visual guide that hierarchically displays MCS proteins and complexes involved in autophagy. In summary, our research has developed a user-friendly visual map for querying, browsing, and visualizing detailed information on autophagy-related MCS proteins and complexes. This tool offers researchers easy access to understand autophagy-related MCS structure, assembly, functions, and therapeutic strategies for related diseases. AutoMCS Navigator is freely available at https://cellknowledge.com.cn/mcsdb/autophagy.html.

Co-occurring mutations in KEAP1 in STK11/LKB1-mutant NSCLC activate NFE2L2/NRF2 to compensate for the loss of STK11-AMPK activity during metabolic adaptation. Characterizing the regulatory crosstalk between the STK11-AMPK and KEAP1-NFE2L2 pathways during metabolic stress is crucial for understanding the implications of co-occurring mutations. Here, we found that metabolic stress increased the expression and phosphorylation of SQSTM1/p62, which is essential for the activation of NFE2L2 and AMPK, synergizing antioxidant defense and tumor growth. The SQSTM1-driven dual activation of NFE2L2 and AMPK was achieved by inducing macroautophagic/autophagic degradation of KEAP1 and facilitating the AXIN-STK11-AMPK complex formation on the lysosomal membrane, respectively. In contrast, the STK11-AMPK activity was also required for metabolic stress-induced expression and phosphorylation of SQSTM1, suggesting a double-positive feedback loop between AMPK and SQSTM1. Mechanistically, SQSTM1 expression was increased by the PPP2/PP2A-dependent dephosphorylation of TFEB and TFE3, which was induced by the lysosomal deacidification caused by low glucose metabolism and AMPK-dependent proton reduction. Furthermore, SQSTM1 phosphorylation was increased by MAP3K7/TAK1, which was activated by ROS and pH-dependent secretion of lysosomal Ca²⁺. Importantly, phosphorylation of SQSTM1 at S24 and S226 was critical for the activation of AMPK and NFE2L2. Notably, the effects caused by metabolic stress were abrogated by the protons provided by lactic acid. Collectively, our data reveal a novel double-positive feedback loop between AMPK and SQSTM1 leading to the dual activation of AMPK and NFE2L2, potentially explaining why co-occurring mutations in STK11 and KEAP1 happen and providing promising therapeutic strategies for lung cancer.Abbreviations: AMPK: AMP-activated protein kinase; BAF1: bafilomycin A₁; ConA: concanamycin A; DOX: doxycycline; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; LN: low nutrient; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NSCLC: non-small cell lung cancer; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PPP2/PP2A: protein phosphatase 2; ROS: reactive oxygen species; PPP3/calcineurin: protein phosphatase 3; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TCL: total cell lysate; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; V-ATPase: vacuolar-type H⁺-translocating ATPase.

Atg9, the only transmembrane protein among many autophagy-related proteins, was first identified in the year 2000 in yeast. Two homologs of Atg9, ATG9A and ATG9B, have been found in mammals. While ATG9B shows a tissue-specific expression pattern, such as in the placenta and pituitary gland, ATG9A is ubiquitously expressed. Additionally, ATG9A deficiency leads to severe defects not only at the molecular and cellular levels but also at the organismal level, suggesting key and fundamental roles for ATG9A. The subcellular localization of ATG9A on small vesicles and its functional relevance to autophagy have suggested a potential role for ATG9A in the lipid supply during autophagosome biogenesis. Nevertheless, the precise role of ATG9A in the autophagic process has remained a long-standing mystery, especially in neurons. Recent findings, however, including structural, proteomic, and biochemical analyses, have provided new insights into its function in the expansion of the phagophore membrane. In this review, we aim to understand various aspects of ATG9 (in invertebrates and plants)/ATG9A (in mammals), including its localization, trafficking, and other functions, in nonneuronal cells and neurons by comparing recent discoveries related to ATG9/ATG9A and proposing directions for future research.Abbreviation: AP-4: adaptor protein complex 4; ATG: autophagy related; cKO: conditional knockout; CLA-1: CLArinet (functional homolog of cytomatrix at the active zone proteins piccolo and fife); cryo-EM: cryogenic electron microscopy; ER: endoplasmic reticulum; KO: knockout; PAS: phagophore assembly site; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SV: synaptic vesicle; TGN: trans-Golgi network; ULK: unc-51 like autophagy activating kinase; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Macroautophagy/autophagy is a conserved pathway allowing the cell to clear and recycle unwanted materials. While decades of research have revealed molecular players and their hierarchical relationships in autophagy, the detailed mechanism by which these molecules function remains largely unknown. In a recent study, Jagan et al. revealed the membrane remodeling ability of two important proteins, MAP1LC3B/LC3B and ATG16L1, in autophagy. LC3B and the ATG12-ATG5-ATG16L1 complex function synergically to induce the formation of phagophore-like membrane cups on membranes both in vitro and in vivo. In addition, the authors showed that the recently characterized C-terminal membrane-binding domain of ATG16L1 is required for the cup formation and the subsequent transition to autophagic vesicles. Together this research provides more insight into the molecular function of LC3B and ATG16L1, as well as a possible mechanism for phagophore biogenesis.