Autophagy最新文献

Macroautophagy/autophagy is best known for its role in maintaining cellular homeostasis through degradation of damaged proteins and organelles. In neurons, autophagy also contributes to the regulation of activity by adjusting the availability of cellular components to physiological demand. In a recent study, we show that autophagy shapes neuronal excitability by restraining a calcium-dependent pathway that couples endoplasmic reticulum calcium release to KCNMA1/BKCa activity at the plasma membrane. When autophagy is lost, this pathway is enhanced, and seizure susceptibility increases.

The inorganic pyrophosphatase PPA2, a matrix-localized protein, maintains mitochondrial function. Here, we identified the role of PPA2 in activating mitochondrial fission signaling. We found that PPA2 overexpression promotes mitochondrial fission by upregulating the mitochondrial translocation of phosphorylated DNM1L S616. Moreover, PPA2 interacts with MTFP1, a mitochondrial inner membrane protein, to induce fission signaling; cells knocked down for MTFP1 and overexpressing PPA2 failed to induce DNM1L activation and subsequent mitochondrial fission. Furthermore, in physiological conditions, PPA2 directed mitochondrial fission at the midzone through MFF-DNM1L, leading to mitochondrial proliferation. Interestingly, during mitochondrial stress following CCCP treatment, PPA2 triggers peripheral fission through FIS1 and DNM1L to segregate parts of damaged mitochondria, which is essential for mitophagy. In addition, PPA2 utilized the C-terminal LC3-interacting region (LIR) of MTFP1 for mitophagy-mediated clearance of damaged mitochondria. In conclusion, PPA2 activates mitochondrial fission signaling through MTFP1-DNM1L and is essential in defining the site of mitochondrial fission, leading to mitochondrial proliferation or mitophagy for maintaining mitochondrial homeostasis.Abbreviations: CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Co-IP: co-immunoprecipitation; CQ: chloroquine; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MLS: mitochondrial localization signal; mtDNA: mitochondrial DNA; OMM: outer mitochondrial membrane; RT: room temperature.

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are two major pathways for maintaining cellular protein homeostasis. Increasing evidence has highlighted the complex interactions and crosstalk between these pathways; however, the specific molecules and mechanisms mediating the interplay between the UPS and autophagy are still not fully elucidated. In this study, we discovered that knocking down the Drosophila Cul2 (Cullin 2)-RING ubiquitin ligase complex adaptor CG12084/DmZer1 impedes autophagy and autophagic flux. DmZer1 interacts with the Drosophila SQSTM1/p62 homolog ref(2)P, promoting its association with ubiquitinated proteins and degradation. ref(2)P is a crucial player in regulating autophagy and the Keap1-cnc/NFE2L2 pathway-mediated antioxidant response. Knockdown of DmZer1 leads to the formation of ref(2)P bodies, which sequester Keap1 and promote cnc/NFE2L2-mediated antioxidant responses under oxidative stress conditions. These findings reveal the pivotal role of DmZer1 in regulating autophagy and the ref(2)P-Keap1-cnc/NFE2L2-mediated oxidative stress response.Abbreviations: ARM: armadillo-like domain; ATG: autophagy related; BTZ: bortezomib; CL1-GFP: GFP fused with a CL1 degron; cnc: cap-n-collar; co-IP: co-immunoprecipitation; CRL2: Cullin 2-RING E3 ubiquitin ligase complex; CQ: chloroquine; CUL2/Cul2: cullin 2; EloB: Elongin B; EloC: Elongin C; esg: escargot; ISCs: intestinal stem cells; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; LLPS: liquid-liquid phase separation; LRR: leucine-rich repeat; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; p-H3: phospho-histone H3; PQ: paraquat; ref(2)P: refractory to sigma P; SQSTM1/p62: sequestosome 1; UBA: ubiquitin-associated; UPS: ubiquitin-proteasome system; VHL: von Hippel-Lindau tumor suppressor; ZER1: zyg-11 related cell cycle regulator.

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.