Autophagy最新文献

The WD40 domain (WDD) of ATG16L1 plays a pivotal role in non-canonical autophagy. This study examined the role of recently identified LAP-like non-canonical autophagy (LNCA) in acute pancreatitis. LNCA involves rapid single-membrane LC3 conjugation to endocytic vacuoles in pancreatic acinar cells. The rationale for this study was the previously observed presence of trypsin in the organelles undergoing LNCA; aberrant trypsin formation is an important factor in pancreatitis development. Here we report that the deletion of WDD (attained in ATG16L1[E230] mice) eliminated LNCA, aggravated caerulein-induced acute pancreatitis and suppressed the fast trypsin degradation observed in both a rapid caerulein-induced disease model and in caerulein-treated isolated pancreatic acinar cells. These experiments indicate that LNCA is a WDD-dependent mechanism and suggest that it plays not an activating but a protective role in acute pancreatitis. Furthermore, palmitoleic acid, another inducer of experimental acute pancreatitis, strongly inhibited LNCA, suggesting a novel mechanism of pancreatic lipotoxicity.Abbreviation: AMY: amylase; AP: acute pancreatitis; CASM: conjugation of Atg8 to single membranes; CCK: cholecystokinin; FAEE model: fatty acid and ethanol model; IL6: interleukin 6; LA: linoleic acid; LAP: LC3-associated phagocytosis; LMPO: lung myeloperoxidase; LNCA: LAP-like non-canonical autophagy; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MPO: myeloperoxidase; PMPO: pancreatic myeloperoxidase; POA: palmitoleic acid; WDD: WD40 domain; WT: wild type.

Long-chain free fatty acids (FFAs) accumulation and oxidative toxicity is a major cause for several pathological conditions. The mechanisms underlying FFA cytotoxicity remain elusive. Here we show that palmitic acid (PA), the most abundant FFA in the circulation, induces S403 phosphorylation of SQSTM1/p62 (sequestosome 1) and its aggregation, which sequesters KEAP1 and activates the non-canonical SQSTM1-KEAP1-NFE2L2 antioxidant pathway. The PA-induced SQSTM1 S403 phosphorylation and aggregation are dependent on SQSTM1 K7-D69 hydrogen bond formation and dimerization in the Phox and Bem1 (PB1) domain, which facilitates the recruitment of TBK1 that phosphorylates SQSTM1 S403. The ubiquitin E3 ligase TRIM21 ubiquitinates SQSTM1 at the K7 residue and abolishes the PB1 dimerization, S403 phosphorylation, and SQSTM1 aggregation. TRIM21 is oxidized at C92, C111, and C114 to form disulfide bonds that lead to its oligomerization and decreased E3 activity. Mutagenizing the three C residues to S (3CS) abolishes TRIM21 oligomerization and increases its E3 activity. TRIM21 ablation leads to decreased SQSTM1 K7 ubiquitination, hence elevated SQSTM1 S403 phosphorylation and aggregation, which confers protection against PA-induced oxidative stress and cytotoxicity. Therefore, TRIM21 is a negative regulator of SQSTM1 phosphorylation, aggregation, and the antioxidant sequestration function. TRIM21 is oxidized to reduce its E3 activity that helps enhance the SQSTM1-KEAP1-NFE2L2 antioxidant pathway. Inhibition of TRIM21 May be a viable strategy to protect tissues from lipotoxicity resulting from long-chain FFAs.Abbreviations: ER: endoplasmic reticulum; FFA: free fatty acid; HMOX1/HO-1: heme oxygenase 1; IB: immunoblotting; IF: immunofluorescence; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; MASH: metabolic dysfunction-associated steatohepatitis; MEF: mouse embryonic fibroblast; NFE2L2/Nrf2: NFE2 like BZIP transcription factor 2; PA: palmitic acid; PB1: Phox and Bem 1; ROS: reactive oxygen species; SLD: steatotic liver disease; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TRIM21: tripartite motif containing 21.

As the central hub of the secretory pathway, the Golgi apparatus plays a crucial role in maintaining cellular homeostasis in response to stresses. Recent studies have revealed the involvement of the Golgi tether, GORASP2, in facilitating autophagosome-lysosome fusion by connecting LC3-II and LAMP2 during nutrient starvation. However, the precise mechanism remains elusive. In this study, utilizing super-resolution microscopy, we observed GORASP2 localization on the surface of autophagosomes during glucose starvation. Depletion of GORASP2 hindered phagophore closure by regulating the association between VPS4A and the ESCRT-III component, CHMP2A. Furthermore, we found that GORASP2 controls RAB7A activity by modulating its GEF complex, MON1A-CCZ1, thereby impacting RAB7A's interaction with the HOPS complex. The assembly of both STX17-SNAP29-VAMP8 and YKT6-SNAP29-STX7 SNARE complexes was also attenuated without GORASP2. These findings suggest that GORASP2 helps seal autophagosomes and activate the RAB7A-HOPS-SNAREs membrane fusion machinery for autophagosome maturation, highlighting its membrane tethering function in response to stresses.Abbreviations: BafA1: bafilomycin A₁; ESCRT: endosomal sorting complex required for transport; FPP: fluorescence protease protection; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GORASP2: golgi reassembly stacking protein 2; GSB: glucose starvation along with bafilomycin A₁; HOPS: homotypic fusion and protein sorting; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PK: proteinase K; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SIM: structured illumination microscopy; UVRAG: UV radiation resistance associated.

TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling.Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.

Genome-wide association studies identified variants around the BIN1 (bridging integrator 1) gene locus as prominent risk factors for late-onset Alzheimer disease. In the present study, we decreased the expression of BIN1 in mouse hippocampal neurons to investigate its neuronal function. Bin1 knockdown via RNAi reduced the dendritic arbor size in primary cultured hippocampal neurons as well as in mature Cornu Ammonis 1 excitatory neurons. The AAV-mediated Bin1 RNAi knockdown also generated a significant regional volume loss around the injection sites at the organ level, as revealed by 7-Tesla structural magnetic resonance imaging, and an impaired spatial reference memory performance in the Barnes maze test. Unexpectedly, Bin1 knockdown led to concurrent activation of both macroautophagy/autophagy and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1). Autophagy inhibition with the lysosome inhibitor chloroquine effectively mitigated the Bin1 knockdown-induced dendritic regression. The subsequent molecular studydemonstrated that increased expression of ULK3 (unc-51 like kinase 3), which is MTOR-insensitive, supported autophagosome formation in BIN1 deficiency. Reducing ULK3 activity with SU6668, a receptor tyrosine kinase inhibitor, or decreasing neuronal ULK3 expression through AAV-mediated RNAi, significantly attenuated Bin1 knockdown-induced hippocampal volume loss and spatial memory decline. In Alzheimer disease patients, the major neuronal isoform of BIN1 is specifically reduced. Our work suggests this reduction is probably an important molecular event that increases the autophagy level, which might subsequently promote brain atrophy and cognitive impairment through reducing dendritic structures, and ULK3 is a potential interventional target for relieving these detrimental effects.Abbreviations: AV: adeno-associated virus; Aβ: amyloid-β; ACTB: actin, beta; AD: Alzheimer disease; Aduk: Another Drosophila Unc-51-like kinase; AKT1: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; AP: autophagosome; BafA1: bafilomycin A₁; BDNF: brain derived neurotrophic factor; BIN1: bridging integrator 1; BIN1-iso1: BIN1, isoform 1; CA1: cornu Ammonis 1; CA3: cornu Ammonis 3; CLAP: clathrin and adapter binding; CQ: chloroquine; DMEM: Dulbecco's modified Eagle medium; EGFP: enhanced green fluorescent protein; GWAS: genome-wide association study; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MRI: magnetic resonance imaging; MTOR; mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; PET: positron emission tomography; qRT-PCR: real-time quantitative reverse transcription PCR; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; TFEB: transcription factor EB; ULK1: unc-51 like kinase 1; ULK3: unc-51 like kinase 3.

Diverse environmental stress factors affect the functionality of proteins and membrane compartments within cells causing potentially irremediable damage to the cell. A major process to eliminate nonfunctional molecular aggregates or damaged organelles under stress conditions is macroautophagy/autophagy, thus making its regulation critical for cellular adaptation and survival. The formation of autophagosomes is coordinated by a wide range of cellular factors and culminates in the closure of the cup-shaped double membrane or phagophore. The endosomal sorting complex required for transport (ESCRT) machinery has been proposed to mediate the sealing of the autophagic membranes. However, the molecular basis for ESCRT recruitment to phagophores under stress conditions are not yet fully understood. We recently described the role of ALIX (ALG-2 interacting protein-X) and its interactor CALB1 (Ca²⁺-dependent Lipid Binding protein 1) in autophagosome maturation during salt stress in Arabidopsis. Our study shows that CALB1 is important for phagophore closure and thus to the subsequent delivery to the vacuole. CALB1 localizes on salt-induced phagophores together with ALIX. CALB1 stimulates the phase separation of ALIX, which can facilitate the further ESCRT recruitment to phagophore membranes.

Cholesterol serves as a vital lipid that regulates numerous physiological processes. Nonetheless, its role in regulating cell death processes remains incompletely understood. In this study, we investigated the role of cholesterol trafficking in immunogenic cell death. Through cell-based drug screening, we identified two antidepressants, sertraline and indatraline, as potent inducers of the nuclear translocation of TFEB (transcription factor EB). Activation of TFEB was mediated through the autophagy-independent lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). Both compounds promoted cholesterol accumulation within lysosomes, resulting in lysosomal membrane permeabilization, disruption of autophagy and cell death that could be reversed by cholesterol depletion. Molecular docking analysis indicated that sertraline and indatraline have the potential to inhibit cholesterol binding to the lysosomal cholesterol transporters, NPC1 (NPC intracellular cholesterol transporter 1) and NPC2. This inhibitory effect might be further enhanced by the upregulation of NPC1 and NPC2 expression by TFEB. Both antidepressants also upregulated PLA2G15 (phospholipase A2 group XV), an enzyme that elevates lysosomal cholesterol. In cancer cells, sertraline and indatraline elicited immunogenic cell death, converting dying cells into prophylactic vaccines that were able to confer protection against tumor growth in mice. In a therapeutic setting, a single dose of each compound was sufficient to significantly reduce the outgrowth of established tumors in a T-cell-dependent manner. These results identify sertraline and indatraline as immunostimulatory agents for cancer treatment. More generally, this research shed light on novel therapeutic avenues harnessing lysosomal cholesterol transport to regulate immunogenic cell death.Abbreviation: ATG5: autophagy related 5; ATG13: autophagy related 13; DKO: double knockout; ICD: immunogenic cell death; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LGALS3: galectin 3; LDL: low-density lipoprotein; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTX: mitoxantrone; NPC1: NPC intracellular cholesterol transporter 1; NPC2: NPC intracellular cholesterol transporter 2; TFE3: transcription factor E3; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1.

HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8^K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8^K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1^P182L and HSPB8^K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8^K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies.

Intervertebral disc degeneration (IVDD) is a leading cause of low back pain that incurs large socioeconomic burdens. Growing evidence reveals that macroautophagy/autophagy dysregulation contributes to IVDD, but the exact role of autophagy and its regulatory mechanisms remain largely unknown. Here, we found that mechanical overloading impaired the autophagic flux of nucleus pulposus (NP) cells in vivo and in vitro. Mechanistically, the impairment of autophagic flux was attributed to lysosomal dysfunction induced by overloading. Overloading could also lead to lysosomal membrane permeabilization and consequent lysosome-dependent cell death. As critical effectors of lysosomal quality control pathways, CHMP4B (charged multivesicular body protein 4B) and TFEB (transcription factor EB) were downregulated in overloading-treated NP cells and degenerative discs. Restoring lysosomal function by CHMP4B or TFEB overexpression attenuated autophagic flux impairment of NP cells and protected against overloading-induced IVDD. Additionally, human IVDD was associated with impaired autophagy, and defective lysosomal quality control was also linked to human IVDD. Collectively, these findings highlighted that lysosomal defects were crucial for mechanical overloading-induced autophagic flux impairment and death of NP cells, suggesting the potential therapeutic relevance of restoring lysosomal function for IVDD.Abbreviations: ADAMTS4: ADAM metallopeptidase with thrombospondin type 1 motif 4; Ad: adenovirus; AO: acridine orange; BafA1: bafilomycin A₁; CHMP4B: charged multivesicular body protein 4B; CTSD: cathepsin D; CV%: coefficient of variation; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complex required for transport; HE: haemotoxylin and eosin; IVDD: intervertebral disc degeneration; LAMP: lysosomal associated membrane protein; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MMP3: matrix metallopeptidase 3; MRI: magnetic resonance imaging; NP: nucleus pulposus; PG: Pfirrmann grade; PI: propidium iodide; RT-qPCR: reverse transcription-quantitative PCR; SOFG: safranin O fast green; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB.

Parkinson disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra, primarily due to mitochondria dysfunction. PRKN (parkin RBR E3 ubiquitin protein ligase) and PINK1 (PTEN induced kinase 1) are linked to early-onset cases of PD and essential for the clearance of damaged mitochondria via selective mitochondrial autophagy (mitophagy). In a recent publication, we detail how a small molecule can activate PRKN mutants that are unable to be phosphorylated, restoring mitophagy in cellular assays. These findings offer hope for the design of therapeutic drugs for some forms of PD.