Autophagy最新文献

{"title":"Mitochondrial protein nmd regulates lipophagy and general autophagy during development.","authors":"Wei Wang, Xufeng Wang, Xiaoqi Zhou, Lu Jiang, Weina Shang, Liquan Wang, Chao Tong","doi":"10.1080/15548627.2025.2522124","DOIUrl":"https://doi.org/10.1080/15548627.2025.2522124","url":null,"abstract":"<p><p>Lipophagy engulfs lipid droplets and delivers them to lysosomes for degradation. We found that lipophagy levels were low in most fly tissues, except for the prothoracic gland (PG) during larval development. Therefore, we performed a small-scale screening in the PG to identify regulators of lipophagy. We discovered that the loss of <i>nmd</i>, a gene encoding a mitochondrial AAA-ATPase, led to developmental failure and reduced lipophagy in the PG. Further studies indicated that <i>nmd</i> was not only required for lipophagy but also essential for general macroautophagy/autophagy in both PG and fat body tissues. Autophagy was induced but blocked at the autophagosome-lysosome fusion stage upon nmd reduction. Additionally, nmd interacted with mitochondrial protein import machinery, such as Tom20, Tom40, and the import cargo, such as Idh. Loss of <i>nmd</i> decreased protein import into mitochondria. Similar to the loss of <i>nmd</i>, reduction of Tom20 or Tom40 also resulted in reduced lipophagy in the PG. In adult flies, reducing <i>nmd</i> expression in the eyes caused lipid droplet accumulation and severe degeneration during aging. Overexpression of bmm, a triglyceride lipase, reduced lipid droplets in the eye but did not rescue the eye degeneration caused by the reduction of <i>nmd</i>.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144499826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Classical swine fever virus hijacks ESCRT-III and VPS4A to promote phagophore closure for accelerating mitophagy.","authors":"Yan Cheng, Yuhang Li, Xiaoqing Bi, Jinxia Chen, Bingqian Zhao, Jishan Bai, Yinbo Ye, Qi Dai, Linke Zou, Jing Chen, Xiuli Feng, Bin Zhou","doi":"10.1080/15548627.2025.2523734","DOIUrl":"https://doi.org/10.1080/15548627.2025.2523734","url":null,"abstract":"<p><p>Classical swine fever virus (CSFV) infection induces complete mitophagy, which is essential for the clearance of damaged mitochondria. The endosomal sorting complex required for transport (ESCRT) machinery plays a vital role in mediating phagophore closure and autophagosome-lysosome fusion during starvation-induced autophagy. Nevertheless, its involvement in CSFV-induced mitophagy and the underlying mechanisms remain insufficiently understood. Here, we found that the ESCRT-III subunits including CHMP1A, CHMP1B, and CHMP4B, along with the AAA-ATPase VPS4, were actively recruited to autophagosomes during CSFV-induced mitophagy. Consistent with this, depletion of CHMP1A, CHMP1B, CHMP4B or VPS4A disrupted mitophagic flux, impairing both PINK1-PRKN-dependent and -independent pathways. Further investigations revealed that CSFV transiently recruited these subunits to nascent autophagosomes for phagophore sealing during mitophagy. Remarkably, multiple CSFV nonstructural proteins (NSPs) including NS3, NS4B, NS5A and NS5B interacted with these ESCRT key subunits and colocalized on mitophagosomes. Taken together, our study identifies CHMP1A, CHMP1B, CHMP4B, and VPS4A as pivotal regulators of phagophore closure in CSFV-induced mitophagy, unveiling novel mechanisms by which the virus manipulates host cellular pathways and highlighting potential therapeutic targets for infection control.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144509953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sindbis virus is suppressed in the yellow fever mosquito aedes aegypti by Atg6/BECN1 (autophagy-related 6)-mediated activation of autophagy.","authors":"Sujit Pujhari, Chan C Heu, Marco Brustolin, Rebecca M Johnson, Donghun Kim, Jason L Rasgon","doi":"10.1080/15548627.2025.2523735","DOIUrl":"10.1080/15548627.2025.2523735","url":null,"abstract":"<p><p>Macroautophagy/autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains relatively underexplored. To address this gap, we use a genetic approach to activate autophagy in the yellow fever mosquito (<i>Aedes aegypti</i>) infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17-amino acid peptide (\"AaBec-1\") derived from the <i>Ae. aegypti Atg6/BECN1</i> (Autophagy-related 6) gene is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of Atg8 and puncta formation. Next, we engineered a recombinant SINV expressing the AaBec-1 peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulates production of infectious virus. The results from this study improve our understanding of the role of autophagy in regulating arbovirus infection in invertebrate hosts and highlight the potential for the autophagy pathway to be exploited for arbovirus control.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144509934","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Excessive autophagic degradation of MYLK3 causes sunitinib-induced cardiotoxicity.","authors":"Ziwei Pan, Lujie Zhu, Xiaochen Wang, Ning Huangfu, Pengpeng Su, Fangkun Yang, Xuyang Fu, Linbin Pu, Qiuli Fu, Jinghai Chen, Hanbin Cui, Ping Liang, Jiaxi Shen","doi":"10.1080/15548627.2025.2524290","DOIUrl":"https://doi.org/10.1080/15548627.2025.2524290","url":null,"abstract":"<p><p>Sunitinib is a receptor tyrosine kinase inhibitor used for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. Clinical data have shown that patients receiving sunitinib develop reduced cardiac function, arrhythmia and heart failure, thereby largely limiting its clinical use. However, the molecular mechanisms underlying sunitinib-induced arrhythmogenesis remain unclear. Here, utilizing the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model, we found that sunitinib caused a variety of deleterious phenotypes, including cardiomyocyte death, sarcomeric disorganization, irregular Ca²⁺ transients, impaired ATP2A2a/SERCA2a (ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2a) activity, arrhythmia, and excessive macroautophagy/autophagy. Mechanistically, SQSTM1/p62 (sequestosome 1) interacts with MYLK3 (myosin light chain kinase 3) and drives excessive autophagic degradation of MYLK3 in sunitinib-treated iPSC-CMs. Downregulation of MYLK3 suppresses the phosphorylation of CAMK2/CAMKII (calcium/calmodulin dependent protein kinase II), thereby reducing the phosphorylation level of its downstream substrate PLN (phospholamban), leading to impaired ATP2A2a/SERCA2a activity and subsequent Ca²⁺ dyshomeostasis and arrhythmia. Moreover, pharmacological intervention of the cardiac myosin activator omecamtiv mecarbil (OM) or overexpression of MYLK3 significantly restored the expression of MYLK3 and reversed pathogenic phenotypes in sunitinib-treated iPSC-CMs. Nanoparticle delivery of OM effectively prevented sunitinib-induced cardiac dysfunction in mice. Our findings suggest that sunitinib-induced MYLK3 degradation causes the inhibition of the CAMK2-PLN-ATP2A2a signaling pathway and leads to sunitinib-induced arrhythmogenesis, and that MYLK3 can act as a novel cardioprotective target for sunitinib-induced cardiotoxicity.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144499825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microaggrephagy: an ESCRT-I-PTPN23-dependent pathway for MAPT/tau aggregate clearance.","authors":"Shoshiro Hirayama, Shigeo Murata","doi":"10.1080/15548627.2025.2525866","DOIUrl":"https://doi.org/10.1080/15548627.2025.2525866","url":null,"abstract":"<p><p>The clearance mechanisms for ubiquitinated protein aggregates, such as MAPT/tau in neurodegenerative diseases, remain incompletely understood, particularly regarding the role of microautophagy. To identify mediators of this process, we performed an unbiased genome-wide CRISPR knockout screen using cells propagating MAPT/tau repeat domain (MAPT/tauRD) aggregates. This screen identified the ESCRT-I complex and the accessory protein PTPN23 as essential for the clearance of ubiquitinated MAPT/tauRD aggregates via a microautophagy-dependent pathway, operating independently of macroautophagy and chaperone-mediated autophagy. We designate this pathway \"microaggrephagy\". Mechanistically, microaggrephagy involves the recognition of polyubiquitinated aggregates by the ESCRT-I subunit TSG101, with PTPN23 acting as an adaptor bridging ESCRT-I and ESCRT-III to facilitate microautophagic engulfment. Furthermore, a disease-associated mutation in the ESCRT-I component UBAP1 disrupts its interaction with PTPN23 and impairs MAPT/tau clearance, implicating dysfunction of this pathway in neurodegenerative pathogenesis. These findings establish microaggrephagy as a distinct cellular mechanism for degrading pathological protein aggregates, provide a molecular basis for its function, and suggest potential therapeutic targets for proteinopathies.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144509933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"NAD⁺ repletion restores cardioprotective autophagy and mitophagy in obesity-associated heart failure by suppressing excessive trophic signaling.","authors":"Mahmoud Abdellatif, Francisco Vasques-Nóvoa, João Pedro Ferreira, Junichi Sadoshima, Abhinav Diwan, Wolfgang A Linke, Guido Kroemer, Simon Sedej","doi":"10.1080/15548627.2025.2522127","DOIUrl":"https://doi.org/10.1080/15548627.2025.2522127","url":null,"abstract":"<p><p>Macroautophagy/autophagy is markedly inhibited in the hearts of elderly obese patients with heart failure and preserved ejection fraction (HFpEF). However, the therapeutic relevance and underlying signaling mechanisms of the decline of autophagy in HFpEF remain unclear. We observed that therapeutic nicotinamide adenine dinucleotide (NAD⁺) repletion via nicotinamide supplementation restores cardioprotective autophagy and mitophagy in preclinical models of obesity-related HFpEF. Targeted and untargeted cardiac acetylome profiling revealed no significant deacetylation of essential autophagy-related proteins, including ATG5, ATG7 and mammalian Atg8-family members (ATG8s), suggesting a SIRT (sirtuin)-independent mechanism of autophagy induction by nicotinamide. Instead, cardiac transcriptomic analysis revealed major shifts in insulin-IGF1 (insulin-like growth factor 1) signaling, a known autophagy inhibitory pathway. Nicotinamide supplementation reversed the HFpEF-associated increase in insulin-IGF1 signaling, whereas exogenous IGF1 counteracts nicotinamide-induced autophagy. Importantly, nicotinamide fails to exert cardioprotective effects in mice lacking the autophagy-related protein ATG5 in cardiomyocytes, implicating autophagy as essential for the therapeutic response. In patients with HFpEF, a metabolic shift diverting nicotinamide away from NAD⁺ biosynthesis toward catabolism strongly correlates with worsening heart failure and increased cardiovascular mortality, even after adjusting for traditional risk factors. In sum, we demonstrate that NAD⁺ replenishment improves cardiometabolic HFpEF by restoring cardiac autophagy through suppression of excessive IGF1 signaling.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144487439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Macrophage <i>TRIM21</i> knockout inhibits septic acute lung injury by downregulating autophagy regulator protein ubiquitination.","authors":"Fei Xie, Jia-Xin Fan, Hao Wang, Qi-Long Song, Yan-Song Xu, Qing-Su Lan, Xiu-Mei Jiang, Jie Cheng, Ya-Min Hou, Hong-Rui Yang, Xu Zhang, Qiu-Ting Zhang, Peng Wang, Long-Hao Liu, Ju-Ying Qian, Wei-Dong Qin, Ming-Xiang Zhang, Jiao-Jiao Pang, Yu-Guo Chen","doi":"10.1080/15548627.2025.2519063","DOIUrl":"10.1080/15548627.2025.2519063","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Acute lung injury (ALI) caused by sepsis is a fatal disease characterized by an systemic inflammatory response to invading pathogens. Inducing macrophage macroautophagy/autophagy is a critical strategy to combat the inflammatory response in septic ALI. The E3 ubiquitin ligase TRIM21 plays important roles in autophagy. However, the mechanism connecting macrophage TRIM21-associated autophagy to ALI development remains unclear. Therefore, this study was aimed to investigate the role of macrophage TRIM21 in septic ALI in human and mice. TRIM21 levels were significantly increased in the macrophages of septic mice and in the peripheral blood mononuclear cells and bronchoalveolar lavage fluid of septic ALI patients relative to the controls. Intriguingly, &lt;i&gt;Trim21&lt;/i&gt;-specific agonist administration exacerbated ALI and inflammatory responses in septic mice. To elucidate the role of macrophage TRIM21 in the development of septic ALI, we developed a clinically relevant macrophage &lt;i&gt;trim21&lt;/i&gt;-specific knockout mouse sepsis model (&lt;i&gt;trim21&lt;/i&gt;&lt;sup&gt;&lt;i&gt;M-KO&lt;/i&gt;&lt;/sup&gt;). &lt;i&gt;trim21&lt;/i&gt; deficiency significantly reduced mortality in septic ALI model mice by inhibiting sepsis-induced pulmonary edema and inflammatory infiltration, thereby improving the mechanical barrier properties of the alveolar mucosal epithelium and permeability of the alveolar membrane. Mechanistically, TRIM21 inhibits macrophage autophagy by enhancing the K11-linked ubiquitination of the autophagy-regulating proteins ULK1, SQSTM1/p62, BECN1/beclin1, and MAP1LC3B/LC3B and accelerating their ubiquitination-dependent proteasome degradation. This further promotes pro-inflammatory M1 macrophage polarization, aggravating the inflammation of septic lung tissue and exacerbating ALI. Collectively, our data demonstrate a novel role for macrophage TRIM21 in mediating autophagy to accelerate septic ALI. These new findings may provide a framework for potential interventions against septic ALI.&lt;b&gt;Abbreviations:&lt;/b&gt; AL: autolysosome; ALI: acute lung injury; ARG1: arginase, liver; ATG12: autophagy related 12; Baf A1: bafilomycin A&lt;sub&gt;1&lt;/sub&gt;; BALF: bronchoalveolar lavage fluid; Bbox: B-box; BMDM: bone marrow-derived macrophages; CC: coiled-coil; CCL2/MCP1: C-C motif chemokine ligand 2; CHX: cycloheximide; CLP: cecum ligation puncture; co-IP: co-immunoprecipitation; CQ: chloroquine; DEG: differentially expressed genes; ELISA: enzyme-linked immunosorbent assay; GO: gene ontology; HE: hematoxylin and eosin; IL1B/IL-1β: interleukin 1 beta; KEGG: Kyoto Encyclopedia of Genes and Genomes; LPS: lipopolysaccharide; 3-MA: 3-methyladenine; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MRC1/CD206: mannose receptor, C type 1; NLRP3: NLR family, pyrin domain containing 3; NOS2/iNOS: nitric oxide synthase 2, inducible; PYCARD/ASC: PYD and CARD domain containing; RNA-seq: RNA-sequencing; RT-qPCR: reverse transcription quantitative PCR; TNF/TNF-α: tumor necrosis factor; ULK1: unc-51 like kinase","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":"1-20"},"PeriodicalIF":0.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum.","authors":"","doi":"10.1080/15548627.2025.2519055","DOIUrl":"https://doi.org/10.1080/15548627.2025.2519055","url":null,"abstract":"","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":"1-2"},"PeriodicalIF":0.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144478230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inhibition of lysosomal LAMTOR1 increases autophagy by suppressing the MTORC1 pathway to ameliorate lipid accumulations in MAFLD.","authors":"Yunyeong Jang, Minjeong Ko, Ju Yeon Lee, Jin Young Kim, Eun-Woo Lee, Ho Jeong Kwon","doi":"10.1080/15548627.2025.2519054","DOIUrl":"https://doi.org/10.1080/15548627.2025.2519054","url":null,"abstract":"<p><p>Metabolic dysfunction-associated fatty liver disease (MAFLD) is a serious metabolic disorder characterized by fat accumulation in the liver, which can trigger liver inflammation and fibrosis, potentially leading to cirrhosis or liver cancer. Despite many studies, effective treatments for MAFLD remain elusive due to its complex etiology. In this study, we have focused on the discovery of therapeutic agents and molecular targets for MAFLD treatment. We demonstrated that the natural compound acacetin (ACA) alleviates MAFLD by regulating macroautophagy/autophagy in a CDAHFD mouse model of rapidly induced steatohepatitis. In addition, ACA inhibits lipid accumulation in 3T3-L1 adipocytes through autophagy induction. To identify the target responsible for the autophagy activity induced by ACA, we performed drug affinity responsive target stability (DARTS) combined with LC-MS/MS proteomic analysis. This led to the identification of LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and MTOR activator 1), a lysosomal membrane adaptor protein. We found that binding of ACA to LAMTOR1 induces its release from the LAMTOR complex, leading to inhibition of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1), thereby increasing autophagy. This process helps ameliorate metabolic disorders by modulating the MTORC1-AMPK axis. Genetic knockdown of LAMTOR1 phenocopies the effects of ACA treatment, further supporting the role of LAMTOR1 as a target of ACA. These findings suggest LAMTOR1 plays a crucial role in ACA's therapeutic effects on MAFLD. In summary, our study identifies LAMTOR1 as a key protein target of ACA, revealing a potential therapeutic avenue for MAFLD by modulating autophagy via the LAMTOR1-MTORC1-AMPK signaling pathway.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144478231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improved detection of lipidated Atg8a by immunoblotting in <i>Drosophila melanogaster</i> cells and tissues enables precise investigation of Atg8a flux and its termination.","authors":"Siri Andresen, Amani Al Outa, Miriam Formica, Jorrit Enserink, Helene Knævelsrud","doi":"10.1080/15548627.2025.2508551","DOIUrl":"10.1080/15548627.2025.2508551","url":null,"abstract":"<p><p>Macroautophagy/autophagy is an essential intracellular catabolic process for maintaining cellular homeostasis. In <i>Drosophila melanogaster</i>, Atg8a lipidation serves as a key marker for autophagy, yet traditional methods often fail to effectively detect its lipidated state. To overcome this limitation, we developed a refined approach that employs N-ethylmaleimide (NEM) to inhibit Atg4, thereby preserving Atg8a lipidation during sample preparation both <i>in vitro</i> and <i>in vivo</i>. We determined the optimal concentration of the autophagic inhibitors bafilomycin A₁ (BafA1) and chloroquine (CQ) required for inhibition of autolysosomal degradation. Furthermore, we investigated the effects of prolonged nutrient deprivation on autophagic flux and TORC1 signaling. Our findings not only validate the effectiveness of this new approach to monitor lipidation of Atg8a but also provide insights into selection of autolysosomal inhibitors and nutrient-dependent regulatory roles of TORC1 in <i>Drosophila</i>.</p>","PeriodicalId":93893,"journal":{"name":"Autophagy","volume":" ","pages":"1-21"},"PeriodicalIF":0.0,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164221","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}