ATG12-ATG5-TECPR1: an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage.

ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12-ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12-ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.

circ_SIRT1 upregulates ATG12 to facilitate Imatinib resistance in CML through interacting with EIF4A3.

Imatinib is the current gold standard for patients with chronic myeloid leukemia (CML). However, the primary and acquired drug resistance seriously limits the efficacy. To identify novel therapeutic target in Imatinib-resistant CML is of crucial clinical significance. CircRNAs have been demonstrated the essential regulatory roles in the progression and drug resistance of cancers. In this study, we identified a novel circRNA (circ_SIRT1), derived from the SIRT1, which is up-regulated in CML. The high expression of circ_SIRT1 is correlated with drug resistance in CML. Knockdown of circ_SIRT1 regulated K562/R cells viability, invasion and apoptosis. Besides, the inhibition of circ_SIRT1 attenuated autophagy level and reduced IC50 to Imatinib of K562/R cells. Mechanistically, circ_SIRT1 directly binds to the transcription factor Eukaryotic Translation Initiation Factor 4A3(EIF4A3) and regulated EIF4A3-mediated transcription of Autophagy Related 12 (ATG12), thereby affecting Imatinib resistance and autophagy level. Overexpression of ATG12 reversed the regulative effects induced by knockdown of circ_SIRT1. Taken together, our findings revealed circ_SIRT1 acted as a potential tumor regulator in CML and unveiled the underlying mechanism on regulating Imatinib resistance. circ_SIRT1 may serve as a novel therapeutic target and provide crucial clinical implications for Imatinib-resistant CML treatment.

Integrated Multi-Omics Analyses Reveal That Autophagy-Mediated Cellular Metabolism Is Required for the Initiation of Pollen Germination.

Autophagy is an evolutionarily conserved mechanism for degrading and recycling various cellular components, functioning in both normal development and stress conditions. This process is tightly regulated by a set of autophagy-related (ATG) proteins, including ATG2 in the ATG9 cycling system and ATG5 in the ATG12 conjugation system. Our recent research demonstrated that autophagy-mediated compartmental cytoplasmic deletion is essential for pollen germination. However, the precise mechanisms through which autophagy regulates pollen germination, ensuring its fertility, remain largely unknown. Here, we applied multi-omics analyses, including transcriptomic and metabolomic approaches, to investigate the downstream pathways of autophagy in the process of pollen germination. Although ATG2 and ATG5 play similar roles in regulating pollen germination, high-throughput transcriptomic analysis reveals that silencing ATG5 has a greater impact on the transcriptome than silencing ATG2. Cross-comparisons of transcriptome and proteome analysis reveal that gene expression at the mRNA level and protein level is differentially affected by autophagy. Furthermore, high-throughput metabolomics analysis demonstrates that pathways related to amino acid metabolism and aminoacyl-tRNA biosynthesis were affected by both ATG2 and ATG5 silencing. Collectively, our multi-omics analyses reveal the central role of autophagy in cellular metabolism, which is critical for initiating pollen germination and ensuring pollen fertility.

The Intrinsically Disordered N Terminus in Atg12 from Yeast Is Necessary for the Functional Structure of the Protein.

The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function. Here, we show that the IDPR in unconjugated Atg12 is positioned in proximity to the UBL domain, a configuration that is important for the functional structure of the protein. A major deletion in the IDPR disrupts intactness of the UBL domain at the unconjugated C terminus, and a mutation in the predicted α0 helix in the IDPR prevents Atg12 from binding to Atg7 and Atg10, which ultimately affects the protein function in the ubiquitin-like conjugation cascade. These findings provide evidence that the IDPR is an indispensable part of the Atg12 protein from yeast.

TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

ARL6IP5 Ameliorates α-Synuclein Burden by Inducing Autophagy via Preventing Ubiquitination and Degradation of ATG12.

Recent advanced studies in neurodegenerative diseases have revealed several links connecting autophagy and neurodegeneration. Autophagy is the major cellular degradation process for the removal of toxic protein aggregates responsible for neurodegenerative diseases. More than 30 autophagy-related proteins have been identified as directly participating in the autophagy process. Proteins regulating the process of autophagy are much more numerous and unknown. To address this, in our present study, we identified a novel regulator (ARL6IP5) of neuronal autophagy and showed that the level of ARL6IP5 decreases in the brain with age and in Parkinson's disease in mice and humans. Moreover, a cellular model of PD (Wild type and A53T mutant α-synuclein overexpression) has also shown decreased levels of ARL6IP5. ARL6IP5 overexpression reduces α-synuclein aggregate burden and improves cell survival in an A53T model of Parkinson's disease. Interestingly, detailed mechanistic studies revealed that ARL6IP5 is an autophagy inducer. ARL6IP5 enhances Rab1-dependent autophagosome initiation and elongation by stabilizing free ATG12. We report for the first time that α-synuclein downregulates ARL6IP5 to inhibit autophagy-dependent clearance of toxic aggregates that exacerbate neurodegeneration.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 µM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1ß (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Vesicle tethering and fusion promoted by LC3/GABARAP proteins is modulated by the ATG12-ATG5-ATG16L1 complex.

Recently, we have examined the membrane anchoring and subsequent lipidation of six members of the LC3/GABARAP protein family, together with their ability to promote membrane tethering and fusion. GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggested the existence of a lipidation threshold as a requisite for tethering and inter-vesicular lipid mixing. The presence of ATG12-ATG5-ATG16L1 (E3 in short) increased and accelerated LC3/GABARAP lipidation and subsequent vesicle tethering. However, E3 hampered LC3/GABARAP capacity to induce inter-vesicular lipid mixing and/or fusion. Our results suggest a model in which, together with the recently described inter-membrane lipid transfer mechanism, LC3/GABARAP could help in the phagophore expansion process through their ability to tether and fuse vesicles. The growing regions would be areas where the LC3/GABARAP proteins could be lipidated in the absence of E3, or else an independent regulatory mechanism would allow lipid/vesicle incorporation and phagophore growth when E3 was present.Abbreviations: Atg/ATG: autophagy-related protein (in yeast/human); E3: ATG12-ATG5-ATG16L1 complex; GABARAP: gamma-aminobutyric acid receptor associated protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3.

Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 µM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.

Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion.

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12-ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.

Circular RNA circPOFUT1 enhances malignant phenotypes and autophagy-associated chemoresistance via sequestrating miR-488-3p to activate the PLAG1-ATG12 axis in gastric cancer.

Circular RNAs are key regulators in regulating the progression and chemoresistance of gastric cancer (GC), suggesting circular RNAs as potential therapeutic targets for GC. The roles of a novel circular RNA circPOFUT1 in GC are unknown. Here, we found that circPOFUT1 was upregulated in GC tissues and cells, and increased circPOFUT1 expression indicated poor prognosis. Overexpression of circPOFUT1 enhanced cell proliferation, migration, invasion and autophagy-associated chemoresistance in GC, which were suppressed by miR-488-3p overexpression. CircPOFUT1 reduced miR-488-3p expression via sponging miR-488-3p in GC cells. PLAG1 interacted with ATG12 and promoted its expression. MiR-488-3p bound to PLAG1 and suppressed the expression of PLAG1 and ATG12 in GC cells. Overexpression of circPOFUT1 enhanced autophagy-associated chemoresistance of GC cells in vivo, but it was inhibited by overexpression of miR-488-3p. Collectively, circPOFUT1 directly sponged miR-488-3p to activate the expression of PLAG1 and ATG12, thus enhancing malignant phenotypes and autophagy-associated chemoresistance in GC. Our findings show the potential of circPOFUT1 as biomarkers and targeting circPOFUT1 as a therapeutic strategy for GC.

LncRNA DDX11-AS1 Promotes Chemoresistance through LIN28A-Mediated ATG12 mRNA Stabilization in Breast Cancer.

INTRODUCTION: During breast cancer chemotherapy, the chemoresistance that frequently accompanies the treatment has become a big challenge. Long noncoding RNAs (LncRNAs) have been related to the development of chemoresistance in multiple cancer types. LncRNA DDX11-AS1 has shown a carcinogenic role in lung and colorectal cancer and was reported to enhance oxaliplatin resistance in gastric cancer and Taxol insensitivity in esophageal cancer. But its role in breast cancer chemotherapy drug resistance remains unknown. This study aimed to investigate the function and mechanism of lncRNA DDX11-AS1 in breast cancer chemoresistance. METHODS: The relationship between DDX11-AS1 and adriamycin (ADR) resistance was confirmed by qPCR, cell viability tests, and survival analysis. Then, RNA immunoprecipitation was conducted to evaluate the interaction between DDX11-AS1 and RNA-binding protein LIN28A. The regulation effect of LIN28A on autophagy-related genes ATG7 or ATG12 was detected by RNA stability assay and Western blot. Their correlation analysis was evaluated in GEO datasets and further validated by immunohistochemical results. The clinical significance of DDX11-AS1, ATG7, or ATG12 was evaluated by Kaplan-Meier Plotter analysis. RESULTS: Here, we reported DDX11-AS1 was significantly upregulated in chemoresistant breast cancer cells and overexpression of DDX11-AS1 promoted ADR resistance in breast cancer. LIN28A could interact with DDX11-AS1 and was involved in DDX11-AS1-mediated ADR resistance. Interfering with LIN28A reversed DDX11-AS1-induced ADR resistance. LIN28A could increase the protein level of ATG7 and ATG12 by increasing their mRNA stability. Survival analysis showed that ATG12 expression level was negatively correlated with the prognosis of breast cancer patients. CONCLUSION: This study clarifies the role of DDX11-AS1 in breast cancer chemoresistance and revealed a new mechanism, that is, interacting with LIN28A to stabilize ATG7 and ATG12 and jointly promote chemorefractory. These findings warrant further in vivo investigations to study DDX11-AS1 as a potential target to overcome chemoresistance.

Apicoplast biogenesis mediated by ATG8 requires the ATG12-ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii.

In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii.Abbreviations: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.

Danhong injection alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy through miRNA-132-3p/ATG12 signal axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI) is a renowned traditional Chinese medicine often used clinically to treat cardiovascular and cerebrovascular diseases. Studies have shown that DHI can significantly alter microRNA (miRNA) expression in the brain tissue. Therefore, exploring specific miRNAs' regulatory mechanisms during treatment with DHI is essential. AIM OF THE STUDY: To investigate DHI's regulatory mechanism on cerebral autophagy in rats with cerebral ischemia-reperfusion injury (CIRI). MATERIAL AND METHODS: Rats were randomly divided into the sham, middle cerebral artery occlusion (MCAO) model, and DHI-treatment groups. The extent of brain damage was evaluated using triphenyl tetrazolium chloride and hematoxylin-eosin staining. Hippocampal cell autophagy was observed using transmission electron microscopy. Autophagy-related proteins were analyzed using western blotting. Differentially expressed miRNAs were screened using high-throughput and real-time quantitative reverse transcription PCR. The relationship between miR-132-3p and ATG12 was confirmed using a dual-luciferase assay. The miR-132-3p mimics and inhibitors were transfected into PC12 cells subjected to oxygen-glucose deprivation (OGD) in vitro and MCAO model rats in vivo. RESULTS: DHI significantly altered the miRNA expression profile in rat brain tissues. The pathological changes in the brain tissues were improved, and the autophagic hippocampal cell vehicles were significantly reduced after DHI treatment. miRNA-132-3p, one of the miRNAs with a significantly different expression, was screened. Kyoto Encyclopedia of Genes and Genomes signal pathway analysis showed that its target genes were closely related to autophagy. Western blotting revealed that the p-PI3K, p-AKT, and mTOR expression increased significantly; AMPK, ULK1, ATG12, ATG16L1, and LC3II/I were downregulated in the DHI group. Dual-luciferase reporter gene experiments showed that miRNA-132-3p could target the ATG12 3'-UTR region directly. In vitro, miRNA-132-3p had a protective effect on OGD/R-induced oxidative stress injury in PC12 cells, improving cell viability, and affecting the expression of autophagy pathway-related proteins. In vivo transfection experiments showed that miR-132-3p could regulate ATG12 expression in CIRI rats' lateral brain tissue, affecting the autophagy signaling pathway. miR-132-3p overexpression reduces CIRI-induced autophagy and protects neurons. CONCLUSION: This study showed that DHI inhibits neuronal autophagy after cerebral ischemia-reperfusion. This may have resulted from miR-132-3p targeting ATG12 and regulating the autophagy signaling pathway protein expression.

ATG5: A central autophagy regulator implicated in various human diseases.

Autophagy, an intracellular conserved degradative process, plays a central role in the renewal/recycling of a cell to maintain the homeostasis of nutrients and energy within the cell. ATG5, a key component of autophagy, regulates the formation of the autophagosome, a hallmark of autophagy. ATG5 binds with ATG12 and ATG16L1 resulting in E3 like ligase complex, which is necessary for autophagosome expansion. Available data suggest that ATG5 is indispensable for autophagy and has an imperative role in several essential biological processes. Moreover, ATG5 has also been demonstrated to possess autophagy-independent functions that magnify its significance and therapeutic potential. ATG5 interacts with various molecules for the execution of different processes implicated during physiological and pathological conditions. Furthermore, ATG5 genetic variants are associated with various ailments. This review discusses various autophagy-dependent and autophagy-independent roles of ATG5, highlights its various deleterious genetic variants reported until now, and various studies supporting it as a potential drug target.

Ginsenoside Rg1 suppresses paraquat-induced epithelial cell senescence by enhancing autophagy in an ATG12-dependent manner.

Paraquat (PQ), as a widely used herbicide, is highly toxic to human. PQ-induced pulmonary fibrosis is the main reason for respiratory failure and death. In PQ-poisoned mice, we find abundant senescent epithelial cells in the lung tissues, which can contribute to the activation of pulmonary fibroblasts. Ginsenoside Rg1 (Rg1), the main active component of ginseng, possess beneficial properties against aging. In our work, we aimed to investigate the potential protective effects of Rg1 on PQ-induced pulmonary fibrosis and the underlying mechanism. In vivo, the treatment of Rg1 can attenuate PQ-induced pulmonary fibrosis and decrease senescence and senescence associated secretory phenotype (SASP) expression. In vitro, Rg1 can effectively eliminate senescent cells via apoptosis, but not normal cells. In addition, we demonstrate that Rg1 can enhance autophagy activity via inducing the expression of ATG12. Inhibition of autophagy via 3-MA or transfection of the siRNA targeting ATG12 can impair the antiaging effect of Rg1. Taken together, our data implicates that Rg1 can protect pulmonary epithelial cells from PQ-induced cellular senescence in an ATG12 dependent manner, which may provide a preventive and therapeutic strategy for PQ poisoning-induced pulmonary fibrosis.

Relish regulates innate immunity via mediating ATG5 activity in Antheraea pernyi.

In innate immunity, autophagy is an important molecular mechanism that plays a critical role in the animal defense system. Given the importance of anti-microbial autophagy in the innate immune processes, the relationship between anti-microbial autophagy and LPS-induced innate immunity in A. pernyi was investigated. Quantitative RT-PCR analysis revealed that autophagy-related genes (ATG6, ATG5, and ATG12) were induced following LPS injection. LPS treatment in the Relish knockdown larvae reduced the expression of autophagy-related genes, especially ATG5. Furthermore, ATG5 depletion decreased the innate immune effect, while its over-expression with ATG12 was induced after the LPS challenge. The dual-luciferase assay revealed that Relish could regulate ATG5 expression by binding directly to the promoter of the ATG5 gene. Overall, our findings show that Relish regulates the ATG5 transcription to eliminate Gram-negative bacteria by anti-microbial autophagy, implying a strong connection between autophagy and innate immunity in immunologic homeostasis.

Evolutionary diversification of the autophagy-related ubiquitin-like conjugation systems.

Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought.Abbreviations: APEAR: ATG8-PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Ornithine decarboxylase functions in both autophagy and apoptosis in response to ultraviolet B radiation injury.

We present a mechanism for how ornithine decarboxylase (ODC) regulates the crosstalk between autophagy and apoptosis. In cancer cells, low-intensity ultraviolet B (UVBL ) induces autophagy while high-intensity UVB (UVBH ) induces apoptosis. Overexpression of ODC decreases UVBL -induced autophagy by inhibiting Atg5-Atg12 conjugation and suppressing the expression of autophagy markers LC3, Atg7, Atg12, and BECN1 proteins. In contrast, when ODC-overexpressing cells are exposed to UVBH radiation, the levels of LC3-II, Atg5-Atg12 conjugate, BECN1, Atg7, and Atg12 increase, while the apoptosis marker cleaved-PARP proteins decrease, indicating that ODC overexpression induced UVBH -induced autophagy but inhibited UVBH -induced cellular apoptosis. Additionally, when exposed to UVBH radiation, silencing BECN1, Atg5, and Atg12 genes results in a decrease in the level of LC3-II proteins but an increase in the level of cleaved-PARP proteins, and apoptotic bodies were significantly increased while autophagosomes were significantly decreased. These findings imply that ODC inhibits apoptosis in cells via the autophagy pathway. The role of Atg12 in ODC-overexpressing cells exposed to UVBH radiation is investigated using site-directed mutagenesis. Our results indicate that the Atg12-D111S mutant has increased cell survival. The Atg12-ΔG186 mutant impairs autophagy and enhances apoptosis. We demonstrate that when ODC-overexpressing cells are silenced for the Atg12 protein, autophagy and apoptosis are strongly affected, and ODC-induced autophagy protects against UVBH -induced apoptosis via the Atg12 protein.

Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers.

Acquired chemotherapy resistance is one of the main culprits in the relapse of breast cancer. But the underlying mechanism of chemotherapy resistance remains elusive. Here, we demonstrate that a small adaptor protein, SH3BGRL, is not only elevated in the majority of breast cancer patients but also has relevance with the relapse and poor prognosis of breast cancer patients. Functionally, SH3BGRL upregulation enhances the chemoresistance of breast cancer cells to the first-line doxorubicin treatment through macroautophagic/autophagic protection. Mechanistically, SH3BGRL can unexpectedly bind to ribosomal subunits to enhance PIK3C3 translation efficiency and sustain ATG12 stability. Therefore, inhibition of autophagy or silence of PIK3C3 or ATG12 can effectively block the driving effect of SH3BGRL on doxorubicin resistance of breast cancer cells in vitro and in vivo. We also validate that SH3BGRL expression is positively correlated with that of PIK3C3 or ATG12, as well as the constitutive occurrence of autophagy in clinical breast cancer tissues. Taken together, our data reveal that SH3BGRL upregulation would be a key driver to the acquired chemotherapy resistance through autophagy enhancement in breast cancer while targeting SH3BGRL could be a potential therapeutic strategy against breast cancer.Abbreviations: ABCs: ATP-binding cassette transporters; Act D: actinomycin D; ACTB/ß-actin: actin beta; ATG: autophagy-related; Baf A1: bafilomycin A1; CASP3: caspase 3; CHX: cycloheximide; CQ: chloroquine; Dox: doxorubicin; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GEO: gene expression omnibus; GFP: green fluorescent protein; G6PD: glucose-6-phosphate dehydrogenase; GSEA: gene set enrichment analysis; IHC: immunochemistry; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3-MA: 3-methyladenine; mRNA: messenger RNA; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; SH3BGRL: SH3 domain binding glutamate-rich protein-like; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.