Autophagy最新文献

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), remains a significant global health challenge. Mtb is transmitted by respiratory aerosols and infects a variety of myeloid populations. Our recent study shows that the Mtb virulence lipid phthiocerol dimycocerosate (PDIM) promotes the intracellular survival of Mtb in macrophages by inhibiting NADPH oxidase, thereby impairing LC3-associated phagocytosis, and in vivo PDIM also antagonizes canonical macroautophagy/autophagy. In addition, mice defective in autophagy in myeloid cells fail to develop B-cell follicles in the lungs during chronic infection. Here, we present a summary of our recent publication, highlighting the most significant findings and discussing how they provide new insight into the role of autophagy and the diversity of lung myeloid cells in the pathogenesis of Mtb.

Synaptic damage is a crucial pathological process in traumatic brain injury. However, the mechanisms driving this process remain poorly understood. In this report, we demonstrate that the accumulation of damaged mitochondria, resulting from impaired mitphagy, plays a significant role in causing synaptic damage. Moreover, copper induced downregulation of BNIP3 is a key player in regulating mitophagy. DMSA alleviates synaptic damage and mitochondrial dysfunction by promoting urinary excretion of copper. Mechanistically, we find that copper downregulate BNIP3 by increasing the nuclear translocation of NFKB, which is triggered by TRIM25-mediated ubiquitination-dependent degradation of NFKBIA. Our study underscores the importance of copper accumulation in the regulation of BNIP3-mediated mitophagy and suggests that therapeutic targeting of the copper-TRIM25-NFKB-BNIP3 axis holds promise to attenuate synaptic damage after traumatic brain injury.

The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.Abbreviation AID: autoinhibitory domain; ATG: autophagy related; CD: catalytic domain; CRD: carbohydrate-recognition domain; CsA: cyclosporin A; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complexes required for transport; GSK3B: glycogen synthase kinase 3 beta; LAMP1: lysosomal associated membrane protein 1; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NT: N-terminal tail; PPP3CB: protein phosphatase 3 catalytic subunit beta; PPP3R1: protein phosphatase 3 regulatory subunit B, alpha; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; VCP/p97: valosin containing protein; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

Prion disease is a fatal and infectious neurodegenerative disorder caused by the trans-conformation conversion of PRNP/PrP^C to PRNP/PrP^Sc. Accumulated PRNP/PrP^Sc-induced ER stress causes chronic unfolded protein response (UPR) activation, which is one of the fundamental steps in prion disease progression. However, the role of various ER-resident proteins in prion-induced ER stress is elusive. This study demonstrated that ARL6IP5 is compensatory upregulated in response to chronically activated UPR in the cellular prion disease model (RML-ScN2a). Furthermore, overexpression of ARL6IP5 overcomes ER stress by lowering the expression of chronically activated UPR pathway proteins. We discovered that ARL6IP5 induces reticulophagy to reduce the PRNP/PrP^Sc burden by releasing ER stress. Conversely, the knockdown of ARL6IP5 leads to inefficient macroautophagic/autophagic flux and elevated PRNP/PrP^Sc burden. Our study also uncovered that ARL6IP5-induced reticulophagy depends on Ca²⁺-mediated AMPK activation and can induce 3 MA-inhibited autophagic flux. The detailed mechanistic study revealed that ARL6IP5-induced reticulophagy involves interaction with soluble reticulophagy receptor CALCOCO1 and lysosomal marker LAMP1, leading to degradation in lysosomes. Here, we delineate the role of ARL6IP5 as a novel ER stress regulator and reticulophagy inducer that can effectively reduce the misfolded PRNP/PrP^Sc burden. Our research opens up a new avenue of selective autophagy in prion disease and represents a potential therapeutic target.Abbreviations: ARL6IP5: ADP ribosylation factor-like GTPase 6 interacting protein 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; CALCOCO1: calcium binding and coiled-coil domain 1; CQ: chloroquine; DAPI: 4'6-diamino-2-phenylindole; ER: endoplasmic reticulum; ERPHS: reticulophagy/ER-phagy sites; KD: knockdown; KD-CON: knockdown control; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MβCD: methyl beta cyclodextrin; 3 MA: 3-methyladenine; OE: overexpression; OE-CON: empty vector control; PrDs: prion diseases; PRNP/PrP^C: cellular prion protein (Kanno blood group); PRNP/PrP^Sc: infectious scrapie misfolded PRNP; Tm: tunicamycin; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

The renoprotective effects of SLC5A2/SGLT2 (solute carrier 5 (sodium/glucose cotransporter), member 2) inhibitors have recently been demonstrated in non-diabetic chronic kidney disease (CKD), even without overt albuminuria. However, the mechanism underlying this renoprotection is largely unclear. We investigated the renoprotective mechanisms of the SLC5A2 inhibitor empagliflozin with a focus on ALB (albumin) reabsorption and macroautophagy/autophagy in proximal tubules using wild-type or drug-inducible lrp2/Megalin or atg5 knockout mice with high-fat diet (HFD)-induced obesity or 5/6 nephrectomy that elevated intraglomerular pressure without overt albuminuria. Empagliflozin treatment of HFD-fed mice reduced several hallmarks of lipotoxicity in the proximal tubules, such as phospholipid accumulation in the lysosome, inflammation and fibrosis. Empagliflozin, which decreases intraglomerular pressure, not only reduced the HFD-induced increase in ALB reabsorption via LRP2 in the proximal tubules (i.e. total nephron ALB filtration), as assessed by urinary ALB excretion caused by genetic ablation of Lrp2, but also ameliorated the HFD-induced imbalance in circulating ALB-bound fatty acids. Empagliflozin alleviated the HFD-induced increase in autophagic demand and successfully prevented autophagic stagnation in the proximal tubules. Similarly, empagliflozin decreased ALB exposure and autophagic demand in 5/6 nephrectomized mice. Finally, empagliflozin reduced HFD-induced vulnerability to ischemia-reperfusion injury, whereas LRP2 blockade and atg5 ablation separately diminished this effect. Our findings indicate that empagliflozin reduces ALB exposure and prevents autophagic stagnation in the proximal tubules even without overt albuminuria. Autophagy improvement may be critical for the renoprotection mediated by SLC5A2 inhibition.

Macroautophagy/autophagyis a lysosomal-regulated degradation process that participates incellular stress and then promotes cell survival or triggers celldeath. Ferroptosis was initially described as anautophagy-independent, iron-regulated, nonapoptotic cell death.However, recent studies have revealed that autophagy is positivelyassociated with sensitivity to ferroptosis. Nonetheless, themolecular mechanisms by which these two types of regulated cell death(RCD) modulate each other remain largely unclear. Here, we screened85 deubiquitinating enzymes (DUBs) and found that overexpression ofUSP13 (ubiquitin specific peptidase 13) could significantlyupregulate NFE2L2/NRF2 (NFE2 like bZIP transcription factor 2)protein levels. In addition, in 39 cases of KRAS-mutated lungadenocarcinoma (LUAD), we found that approximately 76% of USP13overexpression is positively correlated with NFE2L2 overexpression.USP13 interacts with and catalyzes the deubiquitination of thetranscription factor NFE2L2. Additionally, USP13 depletion promotesan autophagy-to-ferroptosis switch invitro andin xenograft tumor mouse models, through the activation of theNFE2L2-SQSTM1/p62 (sequestosome 1)-KEAP1 axis in KRAS mutant cellsand tumor tissues. Hence, targeting USP13 effectively switchedautophagy-to-ferroptosis, thereby inhibiting KRAS (KRASproto-oncogene, GTPase) mutant LUAD, suggesting the therapeuticpromise of combining autophagy and ferroptosis in the KRAS-mutantLUAD.Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; ACTB: actin beta; AL: autolysosomes; AP: autophagosomes; BCL2L1/BCL-xL: BCL2 like 1; CCK8: Cell Counting Kit-8; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; DOX: doxorubicin; DUB: deubiquitinating enzyme; Ferr-1: ferrostatin-1; GPX4: glutathione peroxidase 4; GSEA: gene set enrichment analysis; 4HNE: 4-hydroxynonenal; IKE: imidazole ketone erastin; KEAP1: kelch like ECH associated protein 1; KRAS: KRAS proto-oncogene, GTPase; LCSC: lung squamous cell carcinoma; IF: immunofluorescence; LUAD: lung adenocarcinoma; Lys05: Lys01 trihydrochloride; MAPK1/ERK2/p42: mitogen-activated protein kinase 1; MAPK3/ERK1/p44; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: NFE2 like bZIP transcription factor, 2; NQO1: NAD(P)H quinone dehydrogenase 1; PG: phagophore; RCD: regulated cell death; RAPA: rapamycin; ROS: reactive oxygen species; SLC7A11/xCT: solute carrier family 7 member 11; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBB/beta-tubulin: tubulin, beta; UPS: ubiquitin-proteasome system; USP13: ubiquitin specific peptidase 13.

Type I topoisomerases (TOP1) are critical to remove the topological stress when DNA double strands are unwound. The TOP1 cleavage complexes (TOP1cc) are normally transient, and the stabilization of TOP1cc by its inhibitors, such as camptothecin (CPT), may lead to DNA damage and become cytotoxic. The proteasome pathway degrades trapped TOP1, which is necessary for the repair machinery to gain access to the DNA; however, this process is mainly described when the CPT concentration is high, at levels which are clinically unachievable. In a recently published study, Lascaux et al. identify macroautophagy/autophagy as a new pathway to remove DNA lesions upon clinically relevant low-dose CPT treatment. The autophagy receptor TEX264 binds to TOP1 and brings this protein and its bound DNA fragments to the phagophore; subsequently, they are ultimately delivered to the lysosome for degradation. This study demonstrates the role of autophagy in maintaining genome stability from a new perspective and reveals potential targets to deal with the resistance to TOP1cc inhibitors during cancer treatment.

Cancer cells compensate with increasing mitochondria-derived vesicles (MDVs) to maintain mitochondrial homeostasis, when canonical MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta)-mediated mitophagy is lacking. MDVs promote the transport of mitochondrial components into extracellular vesicles (EVs) and induce tumor metastasis. Although HSP90 (heat shock protein 90) chaperones hundreds of client proteins and its inhibitors suppress tumors, HSP90 inhibitors-related chemotherapy is associated with unexpected metastasis. Herein, we find that HSP90 inhibitor causes mitochondrial damage but stimulates the low LC3-induced MDVs and the release of MDVs-derived EVs. However, why LC3 decreases and what is the transcriptional regulatory mechanism of MDVs formation under HSP90 inhibition remain unknown. Because TFEB (transcription factor EB) is the most important mitophagy transcription factor, and the HSP90 client HCFC1 (host cell factor C1) regulates TFEB transcription, there should be a hidden connection between TFEB, HCFC1 and HSP90 in MDVs formation. Our results support the idea that HSP90 N-terminal inhibition reduces TFEB transcription via decreased HSP90AA1-HCFC1 interaction, which prevents HCFC1 from binding to the TFEB proximal promoter region. Decreased TFEB transcription and consequently reduced LC3, ultimately promoted MDVs formation. Blocking MDVs formation with the microtubule inhibitor nocodazole (NOC) activates the HCFC1-TFEB-LC3 axis, weakens HSP90 inhibitors-induced MDVs and the release of MDVs-derived EVs, inhibits the growth of tumor cell spheres and primary liver tumors, and reduces the extravasation of cancer cells to secondary metastatic sites. Taken together, these data suggest that combination therapy should be used to reduce the metastatic risk of low TFEB-triggered-MDVs formation caused by HSP90 inhibitors.Abbreviation: ACIs: ATP-competitive inhibitors; BaFA1: bafilomycin A1; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CTD: C-terminal domain; EVs: extracellular vesicles; HCFC1: host cell factor C1; HSP90: heat shock protein 90; ILVs: intralumenal vesicles; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MD: middle domain; MDVs: mitochondria-derived vesicles; MQC: mitochondrial quality control; ΔΨm: mitochondrial membrane potential; MVBs: multivesicular bodies; NB: novobiocin; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFs: transcription factors. NOC: nocodazole; NTD: N-terminal nucleotide binding domain; OCR: oxygen consumption rate; RFP: red fluorescent protein; ROS: reactive oxygen species; STA9090: Ganetespib; VPS35: VPS35 retromer complex component.

Clear cell renal cell carcinoma (ccRCC) is tightly associated with VHL (von Hippel-Lindau tumor suppressor) mutation and dysregulated angiogenesis. Accumulating evidence indicates that antiangiogenic treatment abolishing tumor angiogenesis can achieve longer disease-free survival in patients with ccRCC. Atractylenolide I (ATL-I) is one of the main active compounds in Atractylodes macrocephala root extract and exhibits various pharmacological effects, including anti-inflammatory and antitumor effects. In this study, we revealed the potent antitumor activity of ATL-I in ccRCC. ATL-I exhibited robust antiangiogenic capacity by inhibiting EPAS1/HIF2α-mediated VEGFA production in VHL-deficient ccRCC, and it promoted autophagic degradation of EPAS1 by upregulating the ATPase subunit ATP6V0D2 (ATPase H+ transporting V0 subunit d2) to increase lysosomal function and facilitated fusion between autophagosomes and lysosomes. Mechanistically, ATP6V0D2 directly bound to RAB7 and VPS41 and promoted the RAB7-HOPS interaction, facilitating SNARE complex assembly and autophagosome-lysosome fusion. Moreover, ATP6V0D2 promoted autolysosome degradation by increasing the acidification and activity of lysosomes during the later stages of macroautophagy/autophagy. Additionally, we found that ATL-I could decrease the level of EPAS1, which was upregulated in sunitinib-resistant cells, thus reversing sunitinib resistance. Collectively, our findings demonstrate that ATL-I is a robust antiangiogenic and antitumor lead compound with potential clinical application for ccRCC therapy.Abbreviations: ATL-I: atractylenolide I; ATP6V0D2: ATPase H+ transporting V0 subunit d2; CAM: chick chorioallantoic membrane; ccRCC: clear cell renal cell carcinoma; CTSB: cathepsin B; CTSD: cathepsin D; GO: Gene Ontology; HIF-1: HIF1A-ARNT heterodimer; HOPS: homotypic fusion and protein sorting; KDR/VEGFR: kinase insert domain receptor; KEGG: Kyoto Encyclopedia of Genes and Genomes; RCC: renal cell carcinoma; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; TKI: tyrosine kinase inhibitor; V-ATPase: vacuolar-type H±translocating ATPase; VEGF: vascular endothelial growth factor; VHL: von Hippel-Lindau tumor suppressor.