PM2.5 regulates the progression of lung adenocarcinoma through the axis of HCG18, miR-195 and ATG14.

Relevant studies have indicated the association of HCG18 with tumour occurrence and progression. In this study, we observed that PM2.5 can enhance the growth of lung adenocarcinoma cells by modulating the expression of HCG18. Further investigations, including overexpression and knockout experiments, elucidated that HCG18 suppresses miR-195, which in turn upregulates the expression of ATG14, resulting in the upregulation of autophagy. Consequently, exposure to PM2.5 leads to elevated HCG18 expression in lung tissues, which in turn increases Atg14 expression and activates autophagy pathways through inhibition of miR-195, thereby contributing to oncogenesis.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy.

Canonical autophagy is an evolutionarily conserved process that forms double-membrane structures and mediates the degradation of long-lived proteins (LLPs). Noncanonical autophagy (NCA) is an important alternative pathway involving the formation of microtubule-associated protein 1 light chain 3 (LC3)-positive structures that are independent of partial core autophagy proteins. NCA has been defined by the conjugation of ATG8s to single membranes (CASM). During canonical autophagy and NCA/CASM, LC3 undergoes a lipidation modification, and ATG16L1 is a crucial protein in this process. Previous studies have reported that the WDR domain of ATG16L1 is not necessary for canonical autophagy. However, our study found that WDR domain deficiency significantly impaired LLP degradation in basal conditions and slowed down LC3-II accumulation in canonical autophagy. We further demonstrated that the observed effect was due to a reduced interaction between ATG16L1 and FIP200/WIPI2, without affecting lysosome function or fusion. Furthermore, we also found that the WDR domain of ATG16L1 is crucial for chemical-induced NCA/CASM. The results showed that removing the WDR domain or introducing the K490A mutation in ATG16L1 significantly inhibited the NCA/CASM, which interrupted the V-ATPase-ATG16L1 axis. In conclusion, this study highlights the significance of the WDR domain of ATG16L1 for both canonical autophagy and NCA functions, improving our understanding of its role in autophagy.

Calcitriol ameliorates the progression of hepatic fibrosis through autophagy-related gene 16-like 1-mediated autophagy.

BACKGROUND: Calcitriol has the potential to counteract fibrotic diseases beyond its classical action of maintaining calcium and bone metabolism; however, its functional mechanism remains unknown. Autophagy-related gene 16-like 1 (Atg16l1) is one of the genes related to autophagy and is involved in protecting against fibrotic diseases. The present study aimed to explore the contribution of autophagy to the inhibition of calcitriol-induced hepatic fibrosis, as well as its potential molecular mechanism. METHODS: Carbon tetrachloride (Ccl4)-treated mice were established as hepatic fibrosis models and received calcitriol treatment for 6 weeks. Quantification of Sirius red staining and measurement of key fibrotic markers (collagen-1 and α-SMA) was performed to detect hepatic fibrosis. Chloroquine (CQ) treatment was used to observe autophagic flux, and 3-methyladenine (3-MA) was used to inhibit autophagy. Furthermore, the effects of calcitriol on transforming growth factor ß1 (TGFß1)-stimulated primary hepatic stellate cells (HSCs) were detected. Downregulation of Atg16l1 or vitamin D receptor (VDR) in LX-2 cells was used to explore the mechanism of action of calcitriol in fibrosis and autophagy. Additionally, the electrophoretic mobility shift assay (EMSA) was used to investigate the interactions between VDR and ATG16L1. RESULTS: Calcitriol increased the expression of VDR and ATG16L1, enhanced autophagy and attenuated hepatic fibrosis. 3-MA treatment and VDR silencing abolished the protective effects of calcitriol against fibrosis. Calcitriol-induced anti-fibrosis effects were blocked by ATG16L1 suppression. Furthermore, VDR bound to the ATG16L1 promoter and downregulation of VDR decreased the expression of ATG16L1 in LX-2 cells. CONCLUSION: Calcitriol mitigates hepatic fibrosis partly through ATG16L1-mediated autophagy.

DEFB114 protein enhances host resistance to fungal infection through the NOD1/2-ATG16L1-NF-κB signaling pathway.

The overuse of antibiotics has led to the enhanced resistance of many pathogenic bacteria, posing a threat to human health. Therefore, there is a need to develop green and safe alternatives to antibiotics. Beta-defensins play a crucial role in host defense against pathogens and have multifunctional properties, exerting key roles in innate and adaptive immunity, as well as non-immune processes. In this study, a 210 bp long cDNA sequence of yak DEFB114 gene was amplified and successfully expressed in a prokaryotic system. The DEFB114 protein exhibited significant inhibitory effects on the growth of Aspergillus fumigatus in vitro. When co-cultured with yak macrophages, DEFB114 protein enhanced macrophage phagocytic activity and increased nucleic acid fluorescence intensity (P < 0.05). DEFB114 protein also enhanced the activity of yak macrophages stimulated by inactivated Aspergillus fumigatus spores, increased the release of nitric oxide (NO), and promoted the expression of genes such as γ-actin, Lgals, Man2b, and Capg (P < 0.05). In mice experiments, DEFB114 protein promoted resistance against Aspergillus fumigatus infection, by regulating the NOD1/2-ATG16L1-NF-κB pathway to modulate the host immune response and exert its anti-infective effects. In summary, the yak DEFB114 protein could inhibit the growth of Aspergillus fumigatus and enhance the animal's resistance to pathogenic microorganisms, thereby having significant implications in the treatment and prevention of fungal infections.

Rim aperture of yeast autophagic membranes balances cargo inclusion with vesicle maturation.

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

BACKGROUND: Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. METHODS: The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. RESULTS: Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. CONCLUSIONS: Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy in vivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil in vitro and in vivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

Fisetin alleviates cerebral ischemia/reperfusion injury by regulating Sirt1/Foxc1/Ubqln1 pathway-mediated proteostasis.

BACKGROUND: Cerebral ischemia/reperfusion injury (IRI) is pathologically associated with protein damage. The flavonoid fisetin has good therapeutic effects on cerebral IRI. However, the role of fisetin in regulating protein damage during cerebral IRI development remains unclear. This study investigated the pharmacological effects of fisetin on protein damage during cerebral IRI progression and defined the underlying mechanism of action. METHODS: In vivo and in vitro models of cerebral IRI were established by middle cerebral artery occlusion/reperfusion (MACO/R) and oxygen-glucose deprivation/reperfusion (OGD/R) treatment, respectively. Triphenyl tetrazolium chloride staining was performed to detect cerebral infarct size, and the modified neurologic severity score was used to examine neurological deficits. LDH activity and protein damage were assessed using kits. HT22 cell vitality and apoptosis were examined using CCK-8 assay and TUNEL staining, respectively. Interactions between Foxc1, Ubqln1, Sirt1, and Ezh2 were analyzed using CoIP, ChIP and/or dual-luciferase reporter gene assays. RESULTS: Fisetin alleviated protein damage and ubiquitinated protein aggregation and neuronal death caused by MCAO/R and OGD/R. Ubqln1 knockdown abrogated the inhibitory effect of fisetin on OGD/R-induced protein damage, ubiquitinated protein aggregation, and neuronal death in HT22 cells. Further experiments demonstrated that Foxc1 functions as a transcriptional activator of Ubqln1 and that Sirt1 promotes Foxc1 expression by deacetylating Ezh2 and inhibiting its activity. Furthermore, Sirt1 knockdown abrogated fisetin-mediated biological effects on OGD/R-treated HT22 cells. CONCLUSION: Fisetin improved proteostasis during cerebral IRI by regulating the Sirt1/Foxc1/Ubqln1 signaling axis. Our findings strongly suggest that fisetin-mediated inhibition of protein damage after ischemic stroke is a part of the mechanism through which fisetin is neuroprotective in cerebral IRI.

Tepsin binds LC3B to promote ATG9A trafficking and delivery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B preferentially over other members of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.

The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)-ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12-ATG5-ATG16L1 machinery establishes a high level of regulatory control.

A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy.

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 by promoting Atg11 phosphorylation by Atg1 critical for pexophagy during phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits bind not only selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.

The nascent polypeptide-associated complex subunit Egd1 is required for efficient selective mitochondrial degradation in budding yeast.

Selective degradation of dysfunctional or excess mitochondria is a fundamental process crucial for cell homeostasis in almost all eukaryotes. This process relies on autophagy, an intracellular self-eating system conserved from yeast to humans and is thus called mitophagy. Detailed mechanisms of mitophagy remain to be fully understood. Here we show that mitochondrial degradation in budding yeast, which requires the pro-mitophagic protein Atg32, is strongly reduced in cells lacking Egd1, a beta subunit of the nascent polypeptide-associated complex acting in cytosolic ribosome attachment and protein targeting to mitochondria. By contrast, loss of the sole alpha subunit Egd2 or the beta subunit paralogue Btt1 led to only a partial or slight reduction in mitophagy. We also found that phosphorylation of Atg32, a crucial step for priming mitophagy, is decreased in the absence of Egd1. Forced Atg32 hyperphosphorylation almost completely restored mitophagy in egd1-null cells. Together, we propose that Egd1 acts in Atg32 phosphorylation to facilitate mitophagy.

SNX32 is a host restriction factor that degrades African swine fever virus CP204L via the RAB1B-dependent autophagy pathway.

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Exploring the ATG9A interactome uncovers interaction with VPS13A.

ATG9A, a transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid scramblase, and this function is thought to require its interaction with another core autophagy protein, ATG2A, which acts as a lipid transfer protein. Together, ATG9A and ATG2A are proposed to function to expand the growing autophagosome. However, ATG9A is implicated in other pathways including membrane repair and lipid droplet homeostasis. To elucidate other ATG9A interactors within the autophagy pathway, or interactors beyond autophagy, we performed an interactome analysis through mass spectrometry. This analysis revealed a host of proteins involved in lipid synthesis and trafficking, including ACSL3, VPS13A and VPS13C. Furthermore, we show that ATG9A directly interacts with VPS13A and forms a complex that is distinct from the ATG9A-ATG2A complex.

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

KLF15 Transcriptionally Activates ATG14 to Promote Autophagy and Attenuate Damage of ox-LDL-Induced HAECs.

Kruppel-like factor 15 (KLF15) is involved in many cardiovascular diseases and is abnormally expressed in atherosclerosis (AS), but the regulatory mechanism of KLF15 in AS has not been reported so far. RT-qPCR was used to detect the expression of KLF15 and ATG14 in AS patients. Subsequently, human aortic endothelial cells (HAECs) were induced by oxidized low densitylipoprotein (ox-LDL), and the expression of KLF15 in model cells was detected. KLF15 was overexpressed in cells by lipofection transfection, and then CCK8, flow cytometry, Western blot, ELISA, and related assay kits were used to detect cell viability, apoptosis, inflammatory response as well as oxidative stress, respectively. The targeted regulatory relationship between KLF15 and autophagy-related 14 (ATG14) was detected by ChIP and luciferase reporter assays. Following ATG14 silencing in KLF15-overexpressing cells, immunofluorescence and Western blot were used to detect the autophagy. Finally, after the addition of 3-Methyladenine (3-MA), an autophagy inhibitor, the aforementioned experiments were conducted again to further explore the mechanism. The expression of KLF15 and ATG14 were decreased in AS patients and ox-LDL-induced HAECs. Overexpression of KLF15 protected ox-LDL-induced HAECs from damage, which might be achieved through transcriptional regulation of ATG14. In addition, KLF15 could promote autophagy through transcriptional activation of ATG14. KLF15 transcriptionally activated ATG14 to promote autophagy and attenuate damage of ox-LDL-induced HAECs.