Autophagy最新文献

Recently, rapid progress in the field of microautophagy (MI-autophagy) revealed the existence of multiple subtypes that differ in both intracellular membrane dynamics and molecular mechanisms. As a result, a single umbrella term "microautophagy" has become too vague, even creating some confusion among researchers both within and outside the field. We herein describe different subtypes of MI-autophagic processes and propose a systematic approach for naming them more accurately.Abbreviation: ATG, autophagy related; e-MI, endosomal microautophagy; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex required for transport; EV, extracellular vesicle; HSPA8/HSC70, heat shock protein family A (Hsp70) member 8; ILVs, intralumenal vesicles; l-MI, lysosomal microautophagy; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MCOLN1, mucolipin TRP cation channel 1; microautophagy, MI-autophagy; MVBs, multivesicular bodies; SQSTM1, sequestosome 1; v-MI, vacuolar microautophagy.

The mammalian class III phosphatidylinositol-3-kinase complex (PtdIns3K) forms two biochemically and functionally distinct subcomplexes including the ATG14-containing complex I (PtdIns3K-C1) and the UVRAG-containing complex II (PtdIns3K-C2). Both subcomplexes adopt a V-shaped architecture with a BECN1-ATG14 or UVRAG adaptor arm and a PIK3R4/VPS15-PIK3C3/VPS34 catalytic arm. NRBF2 is a pro-autophagic modulator that specifically associates with PtdIns3K-C1 to enhance its kinase activity and promotes macroautophagy/autophagy. How NRBF2 exerts such a positive effect is not fully understood. Here we report that NRBF2 binds to PIK3R4/VPS15 with moderate affinity through a conserved site on its N-terminal MIT domain. The NRBF2-PIK3R4/VPS15 interaction is incompatible with the UVRAG-containing PtdIns3K-C2 because the C2 domain of UVRAG outcompetes NRBF2 for PIK3R4/VPS15 binding. Our crystal structure of the NRBF2 coiled-coil (CC) domain reveals a symmetric homodimer with multiple hydrophobic pairings at the CC interface, which is in distinct contrast to the asymmetric dimer observed in the yeast ortholog Atg38. Mutations in the CC domain that rendered NRBF2 monomeric led to weakened binding to PIK3R4/VPS15 and only partial rescue of autophagy deficiency in nrbf2 knockout cells. In comparison, NRBF2 with its CC domain replaced by a dimeric Gcn4 module showed proautophagic activity comparable to wild type while NRBF2 carrying a tetrameric Gcn4 module showed further enhanced activity. We propose that the oligomeric state of NRBF2 mediated by its CC domain is critical for strengthening the moderate NRBF2-PIK3R4/VPS15 interaction mediated by its MIT domain to fully activate PtdIns3K-C1 and promote autophagy.Abbreviations: ATG: autophagy related; ATG14: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; dCCD: delete CCD; dMIT: delete MIT; Gcn4: general control nonderepressible 4; ITC: isothermal titration calorimetry; IP: immunoprecipitation; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MIM: MIT-interacting motif; MIT: microtubule interacting and trafficking; NMR: nuclear magnetic resonance; NRBF2: nuclear receptor binding factor 2; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WT: wild type.

Acute kidney injury (AKI) is characterized by the dysfunction of renal tubular epithelial cells (TECs), often leading to renal fibrosis. Mitochondrial impairment is a common hallmark across various types of AKI. However, the potential role of circular RNAs (circRNAs) in modulating mitochondrial homeostasis during AKI and subsequent renal fibrosis remains underexplored. Our findings reveal a significant reduction of circAass levels in the renal cortex across all three AKI models. Mechanistically, circAASS mitigates TEC apoptosis and inflammatory responses by promoting mitochondrial homeostasis, thereby attenuating AKI. Specifically, cytoplasmic circAASS acts as a competing endogenous RNA (ceRNA) by sequestering MIR324-3p, which in turn enhances the expression of PINK1, a critical regulator of mitophagy. Additionally, nuclear circAASS directly interacts with the PPARGC1A/PGC-1α protein, inhibiting its ubiquitin-mediated degradation and thereby promoting mitochondrial biogenesis. Furthermore, we demonstrated that the RNA-binding protein IGF2BP2 suppresses circAASS biogenesis by binding to intronic sequences in the AASS pre-mRNA. Restoring circAass in AKI mouse models improves both mitochondrial biogenesis and mitophagy, ameliorating pro-inflammatory responses of TECs and thus mitigating renal fibrosis. Decreased circAASS expression and its association with impaired mitochondrial function in TECs, followed by more severe renal fibrosis, are observed in AKI patients. Collectively, our results suggest that circAASS protects against AKI by regulating mitochondrial homeostasis, highlighting its potential as a therapeutic target for kidney injury.Abbreviations: AAV9: adeno-associated virus serotype 9; AKI: acute kidney injury; BLAST: Basic Local Alignment Search Tool; ceRNA: competing endogenous RNA; circRNA: circular RNA; CKD: chronic kidney disease; CP-AKI: cisplatin-induced AKI; DHE: dihydroethidium; FISH: fluorescence in situ hybridization; HK2: human renal proximal tubular cells; IF: immunofluorescence; H/R: hypoxia-reoxygenation; I/R: ischemia-reperfusion; ISH: in situ hybridization; LPS: lipopolysaccharide; m⁶A: N⁶-methyladenosine; MMP: mitochondrial membrane potential; NC: negative control; ncRNA: non-coding RNA; PAS: periodic acid-schiff staining; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RBPs: RNA-binding proteins; RIP: RNA immunoprecipitation; ROS: reactive oxygen species; RT-qPCR: real-time quantitative polymerase chain reaction; SAKI: septic AKI; Scr: serum creatinine; Seq: sequencing; siRNA: small interfering RNA; TECs: tubular epithelial cells; TEM: transmission electron microscopy.

Diabetic kidney disease (DKD) is a major complication of diabetes, characterized by progressive renal dysfunction and mitochondrial impairment. Mitophagy, a selective form of macroautophagy/autophagy that maintains mitochondrial quality, is essential for kidney homeostasis. However, the molecular mechanisms by which mitophagy links these pathways to DKD remain poorly understood. This study investigated the role of XIAP-ULK1-mediated mitophagy in regulating carnitine metabolism and its therapeutic potential in alleviating DKD. Through a combination of renal biopsy analysis from DKD patients, diabetic mouse models, high-glucose-treated tubular epithelial cells, and molecular docking, we determined that XIAP upregulation led to ULK1 degradation via K48-linked polyubiquitination, impairing mitophagy and disrupting carnitine metabolism. Restoring ULK1 expression through the ULK1 agonist echinacoside and L-carnitine supplementation improved mitophagy and carnitine homeostasis, reducing kidney injury and enhancing mitochondrial function in diabetic mouse models. These findings suggested that targeting the XIAP-ULK1 axis to restore mitophagy and stabilize carnitine metabolism hold significant promise as a therapeutic strategy for DKD, highlighting the importance of metabolic regulation in kidney disease management.Abbreviations: ACR: ALB (albumin):creatinine ratio; AAV: adeno-associated virus; BUN: blood urea nitrogen; CETSA: cellular thermal shift assay; DARTS: drug affinity responsive target stability; DMEM/F12: Dulbecco's modified Eagle medium/nutrient mixture F-12; FBS: fetal bovine serum; HG: high glucose; IHC: immunohistochemistry; IF: immunofluorescence; LC-MS: liquid chromatography-mass spectrometry; MitoQ: Mitoquinone; PCT: proximal convoluted tubule; PPI: protein-protein interaction; PAS: periodic acid-Schiff; RMSD: root mean square deviation; RMSF: root mean square fluctuation; RNA-seq: RNA sequencing; RT-qPCR: reverse transcription quantitative polymerase chain reaction; Scr: serum creatinine; SDH: succinate dehydrogenase; STZ: streptozotocin; TMLHE: trimethyllysine hydroxylase, epsilon; TEM: transmission electron microscopy; TECs: tubular epithelial cells; scRNA-seq: single-cell RNA sequencing; ULK1: unc-51 like autophagy activating kinase 1; XIAP: X-linked inhibitor of apoptosis.

Parkinson disease (PD) and other α-synucleinopathies are characterized by the intracellular aggregates of SNCA/α-synuclein (synuclein, alpha) thought to spread via cell-to-cell transmission. To understand the contributions of various brain cells to the spreading of SNCA pathology, we examined the metabolism of SNCA aggregates in neuronal and glial cells. In neurons, while the full-length SNCA rapidly disappeared following SNCA pre-formed-fibril (PFF) uptake, truncated SNCA accumulated with a half-life of days rather than hours. Epitope mapping and fractionation studies indicate that SNCA fibrils internalized by neurons were truncated at the C-terminal region and remained insoluble. In contrast, microglia and astrocytes rapidly metabolized SNCA fibrils as the half-lives of SNCA fibrils in these glial cells were < 6 h. Differential uptake and processing of SNCA fibrils by neurons and glia was recapitulated in vivo where injection of fluorescently labeled SNCA fibrils initially accumulated in glial cells followed by rapid clearance while neurons stably accumulated SNCA fibrils at a slower rate. Immunolocalization and subcellular fractionation studies show that internalized SNCA PFF was initially localized to endosomes followed by lysosomes. The lysosome was largely responsible for the degradation of internalized SNCA PFF as the inhibition of lysosomal function led to the stabilization of SNCA in all cell types. Significantly, SNCA PFF causes lysosomal dysfunction in neurons. In summary, we show that neurons are inefficient in metabolizing internalized SNCA aggregates, partially because SNCA aggregates cause lysosomal dysfunction, potentially generating aggregation-prone truncated SNCA. In contrast, glial cells may protect neurons from SNCA aggregates by rapidly clearing these aggregates.Abbreviations: 3MA, 3-methyladenine; aa, amino acids; AF, Alexa Fluor; Baf A1, bafilomycin A1; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FL, full-length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMM, high molecular mass; Hs, human; kDa, kilodalton; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; ML, molecular layer; NAC domain, non-amyloidal component; PCN, primary cortical neuron; PD, Parkinson diseases; PFF, pre-formed-fibril; PFF-488, PFF Alexa Fluor-488; PMG, primary microglia; SNCA, synuclein, alpha; SNCA[∆], C-terminally truncated SNCA; SQSTM1/p62, sequestosome 1; TX-100, Triton X-100.

Protein clearance is fundamental to proteome health. In eukaryotes, it is carried out by two highly conserved proteolytic systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Despite their pivotal role, the basal organization of the human protein clearance systems across tissues and cell types remains uncharacterized. Here, we interrogated this organization using diverse bulk and single cell omics datasets. Relative to other protein-coding genes, UPS and ALP genes were more widely expressed, encoded more housekeeping proteins, and were more essential for growth, in accordance with their fundamental roles. Yet, UPS and ALP subsystems had varied expression patterns, and each system showed a layered organization. The smaller layer included genes that were stably and widely expressed across tissues, had elevated expression levels, interacted with more proteins, and were more essential for growth, suggesting that they act as a core. The second larger layer included genes that were differentially expressed across tissues. Tissue-specific upregulation of those genes was associated with tissue-specific functions, phenotypes, and disease susceptibility, as demonstrated computationally and experimentally. Last, we compared protein clearance to other branches of the proteostasis network. Protein clearance and folding were closely coordinated across tissues and more plastic than protein synthesis. Taken together, we propose that the human proteostasis network is organized hierarchically and is tailored to varied proteome compositions. This organization could contribute to and illuminate tissue-selective phenotypes.Abbreviations: ALP: autophagy-lysosome pathway; CDA: chaperone-directed autophagy; DUBs: deubiquitinases; ESCRT: endosomal sorting complexes required for transport; KS: Kolmogorov-Smirnov; MW: Mann-Whitney; TE: tissue-enriched; TS: tissue-specific; UBL: ubiquitin-like protein; UPS: ubiquitin-proteasome system.

The human brain is one of the most metabolically active tissues in the body, due in large part to the activity of trillions of synaptic connections. Under normal conditions, macroautophagy/autophagy at the synapse plays a crucial role in synaptic pruning and plasticity, which occurs physiologically in the absence of disease- or aging-related stressors. Disruption of autophagy has profound effects on neuron development, structure, function, and survival. Neurons are dependent upon maintaining high-quality mitochondria, and alterations in selective mitochondrial autophagy (mitophagy) are heavily implicated in both genetic and environmental etiologies of neurodegenerative diseases. The unique spatial and functional demands of neurons result in differences in the regulation of metabolic, autophagic, mitophagic and biosynthetic processes compared to other cell types. Here, we review recent advances in autophagy and mitophagy research with an emphasis on studies involving primary neurons in vitro and in vivo, glial cells, and iPSC-differentiated neurons. The synaptic functions of genes whose mutations implicate autophagic or mitophagic dysfunction in hereditary neurodegenerative and neurodevelopmental diseases are summarized. Finally, we discuss the diagnostic and therapeutic potentials of autophagy-related pathways.Abbreviations: AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; APP: amyloid beta precursor protein; ASD: autism-spectrum disorder; BDNF: brain-derived neurotrophic factor; BPAN: β-propeller protein associated neurodegeneration; CR: caloric restriction; ΔN111: deleted N-terminal region 111 residues; DLG4/PSD95: discs large MAGUK scaffold protein 4; ER: endoplasmic reticulum; FTD: frontotemporal dementia; HD: Huntington disease; LIR: LC3-interacting region; LRRK2: leucine rich repeat kinase 2; LTD: long-term depression; LTP: long-term potentiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PD: Parkinson spectrum diseases; PGRN: progranulin; PINK1: PTEN induced kinase 1; PRKA/PKA: protein kinase cAMP-activated; PtdIns3P: phosphatidylinositol-3-phosphate; p-S65-Ub: ubiquitin phosphorylated at serine 65; PTM: post-translational modification; TREM2: triggering receptor expressed on myeloid cells 2.

The metabolic co-dependence of the oocyte and surrounding granulosa cells is crucial for oocyte developmental competence. Previous research has shown that serine-glycine and its key downstream metabolites are significantly involved in the process of oocyte maturation. However, the mechanism of serine metabolism and its influence on oocyte maturation remain unclear. In this study, we demonstrate that the serine metabolism enzyme PHGDH, which mediates de novo serine synthesis, is highly activated in granulosa cells and plays a crucial role in maintaining their metabolic and transcriptional homeostasis. By using our previously reported granulosa cell-oocyte co-culture system, we found that macroautophagy/autophagy regulates oocyte maturation by modulating PHGDH-mediated serine metabolism in a stage-specific manner, and this regulation is mediated by CALCOCO2/NDP52-dependent selective autophagy. Additional experiments indicated that S-adenosylmethionine (SAM) is a potential downstream product of serine metabolism, and that restoring SAM significantly rescues both granulosa cell homeostasis and oocyte quality. At the molecular level, we demonstrated that SAM regulates Igf1 expression by altering the H3K4me3 modification level in its promoter region, highlighting a serine-SAM-H3K4me3-Igf1 regulatory axis during oocyte maturation. Finally, we demonstrated that oocyte developmental capacity depends on de novo serine synthesis in granulosa cells during germinal vesicle breakdown (GVBD) stage rather than on the exogenous uptake of serine, and that disruption of serine synthesis significantly affects oocyte developmental capacity. Overall, our findings reveal how serine metabolism links granulosa cells and oocytes, provides new targets for predicting oocyte quality, and may help with strategies for early diagnosis or therapeutic intervention in improving reproductive outcomes.Abbreviations aa: amino acid; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; COCs: cumulus-oocyte complexes; CQ: chloroquine; DEG: differentially expressed gene; GV: germinal vesicle; GVBD: germinal vesicle breakdown; IGF1: insulin-like growth factor 1; MII: metaphase II stage of meiosis; OPTN: optineurin; Pb1: first polar body: PHGDH: 3-phosphoglycerate dehydrogenase; ROS: reactive oxygen species; SAM: s-adenosylmethionine; SQSTM1/p62: sequestosome 1; Ub: ubiquitin; WT: wild-type.

Co-adaptation between viruses and autophagy has equipped viruses with diverse strategies to regulate host redox homeostasis, thereby facilitating viral replication. However, the mechanisms by which viruses manipulate PRDX1 (peroxiredoxin 1), a key antioxidative enzyme, via autophagy remain poorly understood. Here, we demonstrate that infection by Senecavirus A (SVA), an emerging picornavirus, induces PRDX1 degradation, and that PRDX1 negatively regulates viral replication. Decreased PRDX1 expression impairs cellular antioxidant defenses, leading to enhanced reactive oxygen species generation that facilitates SVA replication. Screening of viral proteins revealed that SVA VP1, VP2, and 3A induce PRDX1 degradation through vesicle formation-dependent macroautophagy. Notably, viral VP2 can also recruit HSPA8/HSC70 to specifically target PRDX1, directing it for degradation via LAMP2A-mediated chaperone-mediated autophagy (CMA). Collectively, these findings demonstrate that the SVA VP2 protein plays a central role in orchestrating both macroautophagy- and CMA-mediated PRDX1 degradation, establishing PRDX1 as a potential intervention target for countering SVA infection.Abbreviations: AKT/protein kinase B: AKT serine/threonine kinase; ATP: adenosine triphosphate; BHK-21: baby hamster kidney-21; CAT: catalase; CCCP: BMDMs: bone marrow-derived macrophages; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CQ: chloroquine; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; GPX: glutathione peroxidase; GSH: glutathione; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KO: knockout; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mdivi-1: mitochondrial division inhibitor-1; mM: millimole; MMP: mitochondrial membrane potential; mPTP: mitochondrial permeability transition pore; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetylcysteine; PI3K: phosphoinositide 3-kinase; PRDX1: peroxiredoxin 1; RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction; ROS: reactive oxygen species; SD: standard deviation; SOD: superoxide dismutase; SQSTM1: sequestosome 1; SVA: Senecavirus A; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; μg: microgram; μm: micrometer; μM: micromolar.

Macroautophagy/autophagy protects muscle from proteotoxic stress and maintains tissue homeostasis, yet skeletal muscle relies on it more than most organs. Adult fibers endure constant mechanical strain and require continuous turnover of long-lived proteins, while muscle stem cells (MuSCs) depend on autophagy to remain quiescent, activate after injury, and regenerate effectively. How autophagy is transcriptionally regulated in muscle has been unclear. We identified DEAF1 as a transcriptional brake on autophagy. In MuSCs, DEAF1 controls activation and regeneration and becomes aberrantly elevated with age, promoting protein aggregate formation and cell death. In muscle fibers, DEAF1 is chronically induced during aging, suppressing autophagy and driving functional decline. Exercise reverses DEAF1 induction, restoring autophagy and muscle function. These findings reveal DEAF1 as a key regulator linking autophagy to regeneration and aging, highlighting a therapeutically tractable axis for preserving muscle health.