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 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 transcription via decreased HSP90AA1-HCFC1 interaction, which prevents HCFC1 from binding to the proximal promoter region. Decreased transcription and consequently reduced LC3, ultimately promoted MDVs formation. Blocking MDVs formation with the microtubule inhibitor nocodazole (NOC) activates the HCFC1--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 -triggered-MDVs formation caused by HSP90 inhibitors.: 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.

Exposure of inner mitochondrial membrane resident protein PHB2 (prohibitin 2) during autophagic removal of depolarized mitochondria (mitophagy) depends on the ubiquitin-proteasome system. This uncovering facilitates the PHB2 interaction with phagophore membrane-associated protein MAP1LC3/LC3. It is unclear whether PHB2 is exposed randomly at mitochondrial rupture sites. Prior knowledge and initial screening indicated that VDAC1 (voltage dependent anion channel 1) might play a role in this phenomenon. Through biochemical assays and imaging, we have found that VDAC1-PHB2 interaction increases during mitochondrial depolarization. Subsequently, this interaction enhances the efficiency of PHB2 exposure and mitophagy. To investigate the relevance , we utilized (equivalent to VDAC1) knockout line. Our findings demonstrate that during mitochondrial stress, porin is essential for Phb2 exposure, Phb2-Atg8 interaction and mitophagy. This study highlights that VDAC1 predominantly synchronizes efficient PHB2 exposure through mitochondrial rupture sites during mitophagy. These findings may provide insights to understand progressive neurodegeneration.

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 (Ca-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.

Clear cell renal cell carcinoma (ccRCC) is tightly associated with (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 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.: 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.

Melanosomes play a pivotal role in skin color and photoprotection. In contrast to the well-elucidated pathway of melanosome biogenesis, the process of melanosome degradation, referred to as melanophagy, is largely unexplored. Previously, we discovered that 3,4,5-trimethoxycinnamate thymol ester (TCTE) effectively inhibits skin pigmentation by activating melanophagy. In this study, we discovered a new regulatory signaling cascade that controls melanophagy in TCTE-treated melanocytes. ITCH (itchy E3 ubiquitin protein ligase) facilitates ubiquitination of the melanosome membrane protein MLANA (melan-A) during TCTE-induced melanophagy. This ubiquitinated MLANA is then recognized by an autophagy receptor protein, OPTN (optineurin). Additionally, a phospho-kinase antibody array revealed that TCTE activates PTK2 (protein tyrosine kinase 2), which phosphorylates ITCH, enhancing the ubiquitination of MLANA. Furthermore, inhibition of either PTK2 or ITCH disrupts the ubiquitination of MLANA and the MLANA-OPTN interaction in TCTE-treated cells. Taken together, our findings highlight the critical role of the PTK2-ITCH-MLANA-OPTN cascade in orchestrating melanophagy progression.: α-MSH: alpha-melanocyte-stimulating hormone; dichlone: 2,3-dichloro-1,4-naphthoquinone; ITCH: itchy E3 ubiquitin protein ligase; MITF: melanocyte inducing transcription factor; MLANA: melan-A; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PINK1: PTEN induced kinase 1; PTK2: protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TCTE: 3,4,5-trimethoxycinnamate thymol ester; TPC2: two pore segment channel 2; VDAC1: voltage dependent anion channel 1.

MAVS (mitochondrial antiviral signaling protein) is a crucial adaptor in antiviral innate immunity that must be tightly regulated to maintain immune homeostasis. In this study, we identified the duck TRIM13 (ApdTRIM13) as a novel negative regulator of duck MAVS (ApdMAVS) that mediates the antiviral innate immune response. Upon infection with RNA viruses, ApdTRIM13 expression increased, and it specifically binds to ApdMAVS through its TM domain, facilitating the degradation of ApdMAVS in a manner independent of E3 ligase activity. Furthermore, ApdTRIM13 recruits the autophagic cargo receptor duck SQSTM1 (ApdSQSTM1), which facilitates its interaction with ApdMAVS independent of ubiquitin signaling, and subsequently delivers ApdMAVS to phagophores for degradation. Depletion of ApdSQSTM1 reduces ApdTRIM13-mediated autophagic degradation of ApdMAVS, thereby enhancing the antiviral immune response. Collectively, our findings reveal a novel mechanism by which ApdTRIM13 regulates type I interferon production by targeting ApdMAVS for selective autophagic degradation mediated by ApdSQSTM1, providing insights into the crosstalk between selective autophagy and innate immune responses in avian species.: 3-MA: 3-methyladenine; ATG5: autophagy related 5; baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CARD: caspase recruitment domain; co-IP: co-immunoprecipitation; DEFs: duck embryonic fibroblasts; DTMUV: duck Tembusu virus; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; IKBKE/IKKε: inhibitor of nuclear factor kappa B kinase subunit epsilon; IP: immunoprecipitation; IRF7: interferon regulatory factor 7; ISRE: interferon-stimulated response element; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB: nuclear factor kappa B; pAb: polyclonal antibody; poly(I:C): Polyriboinosinic polyribocytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like-receptor; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious dose; TM: tansmembrane; TOLLIP: toll interacting protein; TRIM: tripartite motif containing; UBA: ubiquitin-associated domain; Ub: ubiquitin; VSV: vesicular stomatitis virus; WT: wild type.

Mitophagy, the process by which cells eliminate damaged mitochondria, is mediated by PINK1 (PTEN induced kinase 1). Our recent research indicates that PINK1 functions as a tumor suppressor in colorectal cancer by regulating cellular metabolism. Interestingly, PINK1 ablation activated the NLRP3 (NLR family pyrin domain containing 3) inflammasome, releasing IL1B (interleukin 1 beta). However, inhibiting the NLRP3-IL1B signaling pathway with an IL1R (interleukin 1 receptor) antagonist or NLRP3 inhibitor did not hinder colon tumor growth after PINK1 loss. To identify druggable targets in PINK1-deficient tumors, ribonucleic acid sequencing analysis was performed on colon tumors from knockout and wild-type mice. Gene Set Enrichment Analysis highlighted the enrichment of iron ion transmembrane transporter activity. Subsequent qualitative polymerase chain reaction and western blot analysis revealed an increase in mitochondrial iron transporters, including mitochondrial calcium uniporter, in PINK1-deficient colon tumor cells and tissues. Live-cell iron staining demonstrated elevated cellular and mitochondrial iron levels in PINK1-deficient cells. Clinically used drugs deferiprone and minocycline reduced mitochondrial iron and superoxide levels, resulting in decreased colon tumor cell growth and . Manipulating the mitochondrial iron uptake protein MCU (mitochondrial calcium uniporter) also affected cell and xenograft tumor growth. This study suggests that therapies aimed at reducing mitochondrial iron levels may effectively inhibit colon tumor growth, particularly in patients with low PINK1 expression.: ANOVA: analysis of variance; APC: adenomatous polyposis coli; cAMP: cyclic adenosine monophosphate; CDX2: caudal type homeobox 2; CGAS: cyclic GMP-AMP synthase; CRC: colorectal cancer; DNA: deoxyribonucleic acid; DFP: deferiprone; DMEM: Dulbecco's modified Eagle medium; DSS: dextran sodium sulfate; ERT2-Cre: Cre recombinase fused to a triple mutant form of the human estrogen receptor; EV: empty vector; GLB: glybenclamide/glyburide; H&E: hematoxylin and eosin; ICP-MS: inductively coupled plasma mass spectrometer; IL1B: interleukin 1 beta; kDa: kilodalton; MCU: mitochondrial calcium uniporter; MKI67: marker of proliferation Ki-67; mRNA: messenger ribonucleic acid; MTT: 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide; NLRP3: NLR family pyrin domain containing 3; OE: overexpression; PBS: phosphate-buffered saline; p-CREB: phosphorylated cAMP responsive element binding protein; PINK1: PTEN induced kinase 1; p-PRKAA/AMPK: phosphorylated protein kinase AMP-activated catalytic subunit alpha; qPCR: qualitative polymerase chain reaction; RNA-seq: ribonucleic acid sequencing; ROS: reactive oxygen species; sg: single guide; sh: short hairpin; SLC25A28: solute carrier family 25 member 28; SLC25A37/MFRN: solute carrier family 25 member 37; STING1: stimulator of interferon response cGAMP interactor 1; TP53/p53: tumor protein p53; TUBA: tubulin alpha; µL: microliter; µm: micrometer; µM: micromolar; mm: millimeter.

PRKN-dependent mitophagy plays a crucial role in maintaining mitochondrial health. Yet, PRKN-deficient mice do not exhibit mitochondrial and cardiac phenotypes at baseline, suggesting the existence of other mitochondrial ubiquitin (Ub) ligases. Here, we discuss our recent work identifying RNF7/RBX2 as a novel mitochondrial Ub ligase. Upon mitochondrial depolarization, RNF7 proteins are recruited to the mitochondria, where they directly ubiquitinate mitochondrial proteins and stabilize PINK1 expression, thereby promoting the clearance of damaged mitochondria and regulating mitochondrial turnover in the heart. The actions of RNF7 in mitochondria do not require PRKN. Ablation of in mouse hearts results in severe mitochondrial dysfunction and heart failure. Our findings demonstrate that RNF7 is indispensable for mitochondrial turnover and cardiac homeostasis. These results open new avenues for exploring new PRKN-independent pathways that regulate mitophagy, which could have significant implications for developing therapeutic interventions for cardiac diseases.

Macroautophagy/autophagy maintains cellular homeostasis by degrading cytoplasmic components and its disruption is linked to Parkinson disease (PD), which is characterized by dopamine depletion and the accumulation of SNCA/α-synuclein aggregates in neurons. Therefore, activation of autophagy is considered a therapeutic strategy for PD; however, autophagy inducers have not yet been developed as therapeutic drugs because they are involved in a wide range of signaling pathways. Here, we focused on the lysosomal clustering around the microtubule-organizing center (MTOC) that can regulate the process of autophagosome-lysosome fusion, the final step of autophagy, and examined how lysosomal clustering affects protein degradation through autophagy. Our study identified six compounds from a high-content screen of 1,200 clinically approved drugs that induce both lysosomal clustering and autophagy. Notably, albendazole reduced SNCA aggregates in a PD model by lysosomal clustering and autophagy. These findings suggest that targeting lysosomal clustering could offer new therapeutic insights for PD.

Mitophagy, the selective autophagic clearance of damaged mitochondria, is considered vital for maintaining mitochondrial quality and cellular homeostasis; however, its molecular mechanisms, particularly under basal conditions, and its role in cellular physiology remain poorly characterized. We recently demonstrated that basal mitophagy is a key feature of primary human cells and is downregulated by immortalization, suggesting its dependence on the primary cell state. Mechanistically, we demonstrated that the PINK1-PRKN-SQSTM1 pathway regulates basal mitophagy, with SQSTM1 sensing superoxide-enriched mitochondria through its redox-sensitive cysteine residues, which mediate SQSTM1 oligomerization and mitophagy activation. We developed STOCK1N-57534, a small molecule that targets and promotes this SQSTM1 activation mechanism. Treatment with STOCK1N-57534 reactivates mitophagy downregulated in senescent and naturally aged donor-derived primary cells, improving cellular senescence(-like) phenotypes. Our findings highlight that basal mitophagy is protective against cellular senescence and aging, positioning its pharmacological reactivation as a promising anti-aging strategy. IR: ionizing radiation; ROS: reactive oxygen species; SARs: selective autophagy receptors.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with the non-neovascular or atrophic form being the most common. Current treatment options are limited, emphasizing the urgent need for new therapeutic strategies. Our key finding is that increased levels of AKT2 in the RPE cells impair lysosomal function and trigger secretory autophagy; a non-canonical macroautophagy/autophagy pathway where cellular materials are released via the plasma membrane rather than being degraded by lysosomes. We showed that this process involves a protein complex, AKT2-SYTL1-TRIM16-SNAP23, releasing factors contributing to drusen biogenesis, a clinical hallmark of AMD development. Importantly, SIRT5 can inhibit this pathway, potentially offering a protective effect. Understanding mechanisms by which this non-canonical autophagy pathway promotes extracellular waste accumulation could provide new insights into drusen biogenesis. Future therapies for atrophic AMD could focus on regulating secretory autophagy or manipulating proteins involved in this process.

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 ULK1 kinase complex plays a crucial role in autophagosome biogenesis. To identify interactors or regulators of ULK1 complex assembly influencing autophagosome biogenesis, we performed an interaction proteomics screen. Employing both affinity purification and proximity labeling of - and -terminal tagged fusion proteins coupled to quantitative mass spectrometry, we identified 317 high-confidence interactors or neighbors of the four ULK1 complex members, including both member-specific and common interactors. Interactions with selective macroautophagy/autophagy receptors indicate the activation of selective autophagy pathways by 90 min of nutrient starvation. Focusing on the ULK1 effector protein BAG2, a common interactor identified by both approaches, we highlight that ULK1 phosphorylates BAG2, supporting the localization of the scaffold and autophagy inducer AMBRA1 to the ER, thereby positively regulating autophagy initiation.: AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; ER: endoplasmic reticulum; HA: hemagglutinin; KD: knockdown; KO: knockout; MS: mass spectrometry; PTM: posttranslational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

All viruses are obligate intracellular parasites that use host machinery to synthesize viral proteins. In infected eukaryotes, viral secreted and transmembrane proteins are synthesized at the endoplasmic reticulum (ER). Many viruses refashion ER membranes into bespoke factories where viral products accumulate while evading host pattern recognition receptors. ER processes are tightly regulated to maintain cellular homeostasis, so viruses must either conform to ER regulatory mechanisms or subvert them to ensure efficient viral replication. Reticulophagy is a catabolic process that directs lysosomal degradation of ER components. There is accumulating evidence that reticulophagy serves as a form of antiviral defense; we call this defense "xERophagy" to acknowledge its relationship to xenophagy, the catabolic degradation of microorganisms by macroautophagy/autophagy. In turn, viruses can subvert reticulophagy to suppress host antiviral responses and support efficient viral replication. Here, we review the evidence for functional interplay between viruses and the host reticulophagy machinery.: AMFR: autocrine motility factor receptor; ARF4: ADP-ribosylation factor 4; ARL6IP1: ADP-ribosylation factor-like 6 interacting protein 1; ATL3: atlastin GTPase 3; ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; BPIFB3: BPI fold containing family B, member 3; CALCOCO1: calcium binding and coiled coil domain 1; CAMK2B: calcium/calmodulin-dependent protein kinase II, beta; CANX: calnexin; CDV: canine distemper virus; CCPG1: cell cycle progression 1; CDK5RAP3/C53: CDK5 regulatory subunit associated protein 3; CIR: cargo-interacting region; CoV: coronavirus; CSNK2/CK2: casein kinase 2; CVB3: coxsackievirus B3; DAPK1: death associated protein kinase 1; DENV: dengue virus; DMV: double-membrane vesicles; EBOV: Ebola virus; EBV: Epstein-Barr Virus; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EMCV: encephalomyocarditis virus; EMV: extracellular microvesicle; ER: endoplasmic reticulum; ERAD: ER-associated degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signalling 1; EV: extracellular vesicle; EV71: enterovirus 71; FIR: RB1CC1/FIP200-interacting region; FMDV: foot-and-mouth disease virus; HCMV: human cytomegalovirus; HCV: hepatitis C virus; HMGB1: high mobility group box 1; HSPA5/BiP: heat shock protein 5; IFN: interferon; IFNG/IFN-γ: interferon gamma; KSHV: Kaposi's sarcoma-associated herpesvirus; LIR: MAP1LC3/LC3-interacting region; LNP: lunapark, ER junction formation factor; MAP1LC3: microtubule-associated protein 1 light chain 3; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAPK/JNK: mitogen-activated protein kinase; MeV: measles virus; MHV: murine hepatitis virus; NS: non-structural; PDIA3: protein disulfide isomerase associated 3; PRR: pattern recognition receptor; PRRSV: porcine reproductive and respiratory syndrome virus; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RHD: reticulon homology domain; RTN3: reticulon 3; RTN3L: reticulon 3 long; sAIMs: shuffled Atg8-interacting motifs; SARS-CoV: severe acute respiratory syndrome coronavirus; SINV: Sindbis virus; STING1: stimulator of interferon response cGAMP interactor 1; SVV: Seneca Valley virus; SV40: simian virus 40; TEX264: testis expressed gene 264 ER-phagy receptor; TFEB: transcription factor EB; TRAF2: TNF receptor-associated factor 2; UIM: ubiquitin-interacting motif; UFM1: ubiquitin-fold modifier 1; UPR: unfolded protein response; VAPA: vesicle-associated membrane protein, associated protein A; VAPB: vesicle-associated membrane protein, associated protein B and C; VZV: varicella zoster virus; WNV: West Nile virus; XBP1: X-box binding protein 1; XBP1s: XBP1 spliced; xERophagy: xenophagy involving reticulophagy; ZIKV: Zika virus.

Extensive interconnection has been established between clathrin-mediated endocytosis (CME) and the macroautophagy/autophagy pathway in yeast and mammals. However, the evidence that connects these two pathways in plants has been limited. Starting from the phenotypic similarities in carbon starvation and immune responses shared between the double mutant of CLC2 (clathrin light chain 2) and , , and the mutant in Arabidopsis, we found that the autophagy pathway is compromised in the mutant. Subsequently, we demonstrated that CLC2 interacts specifically with ATG8h and ATG8i, two clade II ATG8 isoforms. The CLC2-ATG8h/ATG8i interaction depends on an Atg8-family interacting motif (AIM) present in CLC2 and an AIMs docking site (ADS) present in ATG8h, respectively. In addition, CLC2-GFP is subjected to autophagic degradation and the degradation of GFP-ATG8h is significantly reduced in the mutant. Last, simultaneously knocking out and enhances disease resistance, corroborating the functional relevance of the CLC2-ATG8h/8i interactions. These findings reveal that CME and the autophagy pathway are intersected via CLC2-ATG8h/8i interactions in Arabidopsis.

Prion disease is a fatal and infectious neurodegenerative disorder caused by the trans-conformation conversion of PRNP/PrP to PRNP/PrP. Accumulated PRNP/PrP-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 burden by releasing ER stress. Conversely, the knockdown of ARL6IP5 leads to inefficient macroautophagic/autophagic flux and elevated PRNP/PrP burden. Our study also uncovered that ARL6IP5-induced reticulophagy depends on Ca-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 burden. Our research opens up a new avenue of selective autophagy in prion disease and represents a potential therapeutic target.: 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: cellular prion protein (Kanno blood group); PRNP/PrP: infectious scrapie misfolded PRNP; Tm: tunicamycin; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

GJA1/Cx43 (gap junction protein alpha 1) has long been associated with gap junctions-mediated communication between adjacent cells. However, recent data have defied this concept, with studies implicating GJA1 in other biological processes, such as macroautophagy/autophagy regulation, mitochondrial activity and extracellular vesicles biology. In our recent study we unveiled an additional role played by GJA1 in lysosomal trafficking. We demonstrate that GJA1 promotes the exocytosis of damaged lysosomes, through a mechanism that relies on ACTR2/ARP2-ACTR3/ARP3-dependent actin remodeling. Our findings ascribe to GJA1 an important role during pathogen infection and lysosomal storage disorders, favoring the release of dysfunctional lysosomes.

Protein homeostasis (proteostasis) refers to the balance of the cellular protein environment, tightly regulated by pathways governing protein synthesis, folding, trafficking, and degradation. Growing evidence supports the interconnection of these pathways to ensure the robustness of the proteo-stasis network. A recent study by Park et al. showed that, in macroautophagy/autophagy-deficient cells, the loss of proteasome or nuclear pore components causes synthetic lethality, as cytoplasmic proteins that accumulate under impaired autophagy are transported to the nucleus and degraded by nuclear proteasomes. The authors illustrated the mechanistic basis for why cells with conditions such as Huntington disease, where both autophagy and cytoplasm-to-nuclear shuttling are compromised, are more vulnerable to proteostasis perturbation.: 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 or 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 LRP2 in the proximal tubules (. total nephron ALB filtration), as assessed by urinary ALB excretion caused by genetic ablation of , 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 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.

The plasma concentration of the macroautophagy/autophagy inhibitor DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) increases with aging and body mass index (BMI). Both advanced age and obesity are among the most important risk factors for the development of cancer. We observed that patients with cancer predisposition syndromes due to mutations in , and exhibit abnormally high plasma DBI/ACBP levels. Additionally, patients without known cancer predisposition syndromes also manifest higher DBI/ACBP levels before imminent cancer diagnosis (within 0-3 years) as compared to age and BMI-matched controls who remain cancer-free. Thus, supranormal plasma DBI/ACBP constitutes a risk factor for later cancer development. Mouse experimentation revealed that genetic or antibody-mediated DBI/ACBP inhibition can delay the development or progression of cancers. In the context of chemoimmunotherapy, DBI/ACBP neutralization enhances tumor infiltration by non-exhausted effector T cells but reduces infiltration by regulatory T cells. This resulted in better cancer control in models of breast cancer, non-small cell lung cancer and sarcoma. We conclude that DBI/ACBP constitutes an actionable autophagy checkpoint for improving cancer immunosurveillance. : BMI, body mass index; CTL, cytotoxic T lymphocyte; DBI, diazepam binding inhibitor, acyl-CoA binding protein; mAb, monoclonal antibody; NSCLC, non-small cell lung cancer; PDCD1/PD-1, programmed cell death 1; scRNA-seq, single-cell RNA sequencing; T, regulatory T cell.