The emerging significance of Vac8, a multi-purpose armadillo-repeat protein in yeast.

Vac8 is the sole armadillo-repeat (ARM) protein in yeast. The function of Vac8 in the cytoplasm-to-vacuole targeting pathway has been known for a long time but its role in the phagophore assembly site localization and recruitment of autophagy-related protein complexes is slowly coming to light. Because Vac8 is also involved in formation of the nuclear-vacuole junction and vacuole inheritance, the protein needs to be a competent and wide-ranging mediator of cellular processes. In this article, we discuss two recent studies reporting on Vac8 and its binding partners. We describe Vac8 in the context of crystallized protein complexes as well as predicted models to reveal the versatility of Vac8 and its potential to become a subject of future autophagy research.Abbreviation: ARM, armadillo repeat; Cvt, cytoplasm-to-vacuole targeting; IDPR, intrinsically disordered protein region NVJ, nucleus-vacuole junction; SEC, size-exclusion chromatography.

Sodium fluoride-induced autophagy of ameloblast-like cells via the p-ULk1/ATG13/LC3B pathway in vitro.

OBJECTIVE: To investigate the effects of sodium fluoride on the ameloblast and reveal the mechanism of dental fluorosis. MATERIALS AND METHODS: Mouse ameloblast-like cell line (ALC) cells were treated with various concentrations of NaF, and subjected to Incucyte, fluorescence immunoassay, transmission electron microscopy, reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot for autophagy examination, alkaline phosphatase and alizarin red staining for mineralization after osteogenic induction. RESULTS: NaF exerts a dose-dependent inhibitory effect on ALC cell growth. TEM and fluorescence immunoassay showed that 1.5 mM or higher concentrations of NaF could induce a fusion of lysosome and mitochondria, finally increasing the number of autophagosome. RT-qPCR and western blot showed that the upregulation of autophagy related gene 13 (ATG13), downregulation of phosphorylated Unc-51-like kinase 1 (p-ULK1) were found in NaF-induced autophagy of ALC cells. The knockdown of ATG13 could rescue it as well as the expression of p-ULK1 and LC3B. Besides, alizarin red staining showed that fluoride under these concentrations could promote the mineralization of ALC. CONCLUSIONS: The data show that fluoride in higher concentration can induce autophagy via the p-ULk1/ATG13/LC3B pathway of ALCs than lower ones promote mineralization in vitro, which provides insight into the function of NaF in the autophagy and mineralization of ameloblast.

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.

The role of the HORMA domain proteins ATG13 and ATG101 in initiating autophagosome biogenesis.

Autophagy is a process of regulated degradation. It eliminates damaged and unnecessary cellular components by engulfing them with a de novo-generated organelle: the double-membrane autophagosome. The past three decades have provided us with a detailed parts list of the autophagy initiation machinery, have developed important insights into how these processes function and have identified regulatory proteins. It is now clear that autophagosome biogenesis requires the timely assembly of a complex machinery. However, it is unclear how a putative stable machine is assembled and disassembled and how the different parts cooperate to perform its overall function. Although they have long been somewhat enigmatic in their precise role, HORMA domain proteins (first identified in Hop1p, Rev7p and MAD2 proteins) autophagy-related protein 13 (ATG13) and ATG101 of the ULK-kinase complex have emerged as important coordinators of the autophagy-initiating subcomplexes. Here, we will particularly focus on ATG13 and ATG101 and the role of their unusual metamorphosis in initiating autophagosome biogenesis. We will also explore how this metamorphosis could potentially be purposefully rate-limiting and speculate on how it could regulate the spontaneous self-assembly of the autophagy-initiating machinery.

Mec1-Rad53 Signaling Regulates DNA Damage-Induced Autophagy and Pathogenicity in Candida albicans.

DNA damage activates the DNA damage response and autophagy in C. albicans; however, the relationship between the DNA damage response and DNA damage-induced autophagy in C. albicans remains unclear. Mec1-Rad53 signaling is a critical pathway in the DNA damage response, but its role in DNA damage-induced autophagy and pathogenicity in C. albicans remains to be further explored. In this study, we compared the function of autophagy-related (Atg) proteins in DNA damage-induced autophagy and traditional macroautophagy and explored the role of Mec1-Rad53 signaling in regulating DNA damage-induced autophagy and pathogenicity. We found that core Atg proteins are required for these two types of autophagy, while the function of Atg17 is slightly different. Our results showed that Mec1-Rad53 signaling specifically regulates DNA damage-induced autophagy but has no effect on macroautophagy. The recruitment of Atg1 and Atg13 to phagophore assembly sites (PAS) was significantly inhibited in the mec1Δ/Δ and rad53Δ/Δ strains. The formation of autophagic bodies was obviously affected in the mec1Δ/Δ and rad53Δ/Δ strains. We found that DNA damage does not induce mitophagy and ER autophagy. We also identified two regulators of DNA damage-induced autophagy, Psp2 and Dcp2, which regulate DNA damage-induced autophagy by affecting the protein levels of Atg1, Atg13, Mec1, and Rad53. The deletion of Mec1 or Rad53 significantly reduces the ability of C. albicans to systematically infect mice and colonize the kidneys, and it makes C. albicans more susceptible to being killed by macrophages.

Site-specific Atg13 methylation-mediated autophagy regulates epithelial inflammation in PM2.5-induced pulmonary fibrosis.

Fine particulate matters (PM2.5) increased the risk of pulmonary fibrosis. However, the regulatory mechanisms of lung epithelium in pulmonary fibrosis remained elusive. Here we developed PM2.5-exposure lung epithelial cells and mice models to investigate the role of autophagy in lung epithelia mediating inflammation and pulmonary fibrosis. PM2.5 exposure induced autophagy in lung epithelial cells and then drove pulmonary fibrosis by activation of NF-κB/NLRP3 signaling pathway. PM2.5-downregulated ALKBH5 protein expression promotes m6A modification of Atg13 mRNA at site 767 in lung epithelial cells. Atg13-mediated ULK complex positively regulated autophagy and inflammation in epithelial cells with PM2.5 treatment. Knockout of ALKBH5 in mice further accelerated ULK complex-regulated autophagy, inflammation and pulmonary fibrosis. Thus, our results highlighted that site-specific m6A methylation on Atg13 mRNA regulated epithelial inflammation-driven pulmonary fibrosis in an autophagy-dependent manner upon PM2.5 exposure, and it provided target intervention strategies towards PM2.5-induced pulmonary fibrosis.

ATG13 is involved in immune response of pathogen invasion in blood clam Tegillarca granosa.

Mammalian autophagy-related gene 13 (ATG13) is a vital component of the ATG1 autophagy initiation complex which plays an essential role in autophagy. However, the molecular function of ATG13 in pathogen defense in invertebrates is still poorly understood. In this study, the full-length cDNA sequence of blood clam Tegillarca granosa ATG13 (TgATG13) was obtained, which was 1,918 bp in length, including 283 bp 5' UTR, 252 bp 3' UTR and 1,383 bp open reading frame (ORF) encoding 460 amino acids. Phylogenetic analysis revealed that TgATG13 had the closest relationship with that of Crassostrea Virginica. Quantitative real-time PCR results showed that the transcript of TgATG13 was universally expressed in various tissues of blood clam, with the highest expression level in hemocytes. The expression level of TgATG13 was robustly increased after exposure of both Vibrio alginolyticus and LPS. Fluorescence confocal microscopy further showed that TgATG13 promoted the production of autophagosome. In summary, our study demonstrated that TgATG13 was involved in the immune regulation of blood clam during pathogen invasion, deepening our understanding of the innate immune mechanism of blood clam.

Mec1 regulates PAS recruitment of Atg13 via direct binding with Atg13 during glucose starvation-induced autophagy.

Mec1 is a DNA damage sensor, which performs an essential role in the DNA damage response pathway and glucose starvation-induced autophagy. However, the functions of Mec1 in autophagy remain unclear. In response to glucose starvation, Mec1 forms puncta, which are recruited to mitochondria through the adaptor protein Ggc1. Here, we show that Mec1 puncta also contact the phagophore assembly site (PAS) via direct binding with Atg13. Functional analysis of the Atg13-Mec1 interaction revealed two previously unrecognized protein regions, the Mec1-Binding Region (MBR) on Atg13 and the Atg13-Binding Region (ABR) on Mec1, which mediate their mutual association under glucose starvation conditions. Disruption of the MBR or ABR impairs the recruitment of Mec1 puncta and Atg13 to the PAS, consequently blocking glucose starvation-induced autophagy. Additionally, the MBR and ABR regions are also crucial for DNA damage-induced autophagy. We thus propose that Mec1 regulates glucose starvation-induced autophagy by controlling Atg13 recruitment to the PAS.

MALT-1 shortens lifespan by inhibiting autophagy in the intestine of C. elegans.

The caspase-like protease MALT1 promotes immune responses and oncogenesis in mammals by activating the transcription factor NF-κB. MALT1 is remarkably conserved from mammals to simple metazoans devoid of NF-κB homologs, like the nematode C. elegans. To discover more ancient, NF-κB -independent MALT1 functions, we analysed the phenotype of C. elegans upon silencing of MALT-1 expression systemically or in a tissue-specific manner. MALT-1 silencing in the intestine caused a significant increase in life span, whereas intestinal overexpression of MALT-1 shortened life expectancy. Interestingly, MALT-1-deficient animals showed higher constitutive levels of autophagy in the intestine, which were particularly evident in aged or starved nematodes. Silencing of the autophagy regulators ATG-13, BEC-1 or LGG-2, but not the TOR homolog LET-363, reversed lifespan extension caused by MALT-1 deficiency. These findings suggest that MALT-1 limits the lifespan of C. elegans by acting as an inhibitor of an early step of autophagy in the intestine.

bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

Autophagy regulation of ATG13 and ATG27 on biofilm formation and antifungal resistance in Candida albicans.

Autophagy is a highly conserved catabolic pathway that is vital for cells; however, the effects of autophagy on the biofilm formation and antifungal resistance of Candida albicans are still unknown. In this study, the potential molecular mechanisms of autophagy in biofilm formation and antifungal resistance were investigated. It was found that 3536 genes were differentially expressed between biofilm and planktonic C. albicans. ATG gene expression and autophagy activity were higher in biofilm than in planktonic C. albicans. Autophagic activities were higher in matured biofilms than that in pre-matured biofilms. Autophagy was involved in C. albicans biofilm formation and its activity increased during biofilm maturation. Further, ALP activity, AO staining cells, and autophagosomes inside cells were obviously reduced in biofilms of atg13Δ/Δ and atg27Δ/Δ strains; moreover, biofilm formation and antifungal resistance were also significantly decreased. Lastly, autophagy regulates biofilm formation and drug resistance of C. albicans and could be served as a new molecular target to the C. albicans biofilm infections.

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy.

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores.

The effect of lactoferrin on ULK1 and ATG13 genes expression in breast cancer cell line MCF7 and bioinformatics studies of protein interaction between lactoferrin and the autophagy initiation complex.

Recently, the study of autophagy and its mechanism on the cancer cell growth process has received much attention. lactoferrin (Lf) is a glycoprotein with various biological activities, including antibacterial, antiviral, anti-cancer, etc. In the present study, the effect of different concentrations of lactoferrin on the expression of ULK1 and ATG13 genes was evaluated in breast cancer cell line MCF7 using real-time PCR technique as well as the molecular mechanism of these two genes and their proteins in the autophagy pathway and the relationship between lactoferrin and these proteins were investigated by bioinformatics studies. The result showed that the expression of the ULK1 gene at a concentration of 500 µg/ml of lactoferrin was significantly (P < 0.007) increased compared to the control and two other concentrations. Also, the expression of the ATG13 gene at all three concentrations was not significantly different from each other and compared to the control (P = 0.635). In the immunoblot of ULK1 protein at a concentration of 500 µg, more protein expression was observed. The binding mode of lactoferrin with ULK1, ATG13, and ATG101 proteins was obtained using docking. According to docking results, the N-lobe region of lactoferrin interacts with the PS domain of the ULK1 protein, and the N-lobe region of lactoferrin interacts with the horma domain of the ATG 13 and ATG101 proteins. The results show that lactoferrin, in addition to acting on the gene, interacts with ULK1, ATG13, and ATG101 proteins. Since all three proteins are components of the autophagy initiation complex, lactoferrin can induce autophagy in this way.

Beclin1- and Atg13-dependent autophagy activation and morroniside have synergistic effect on osteoblastogenesis.

Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the in vivo effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.

Cdc14 plans autophagy for meiotic cell divisions.

The role of meiotic proteasome-mediated degradation has been extensively studied. At the same time, macroautophagy/autophagy only emerged recently as an essential regulator for meiosis progression. Our recent publication showed that autophagy in meiotic cells exhibits a temporal pattern distinct from that in quiescent cells or mitotic cells under prolonged starvation. Importantly, autophagic activity oscillates during meiotic cell divisions, i.e., meiosis I and meiosis II, which can accelerate meiotic progression and increase sporulation efficiency. Our in vitro and in vivo assays revealed that the conserved phosphatase Cdc14 stimulates autophagy initiation during meiotic divisions, specifically in anaphase I and II, when a subpopulation of active Cdc14 relocates to the cytosol and interacts with phagophore assembly sites (PAS) triggering the dephosphorylation of Atg13 to stimulate Atg1 kinase activity and autophagy. Together, our findings reveal a mechanism for the coordination of autophagy activity in the context of meiosis progression.

Mapping the proximity interactome of ATG9A reveals unexpected dynamics of ULK1 complex proteins.

ATG9A is essential for macroautophagy/autophagy and considered to be one of the earliest ATG (autophagy related) proteins recruited to sites of autophagosome biogenesis. Recent data suggest ATG9A vesicles may even form the lipid seed of the autophagosome. However, ATG9A regulation is still poorly understood, which is likely at least partly due to challenges inherent to studying an intracellular transmembrane protein with no apparent enzymatic activity. To help overcome these challenges, we used BioID and quantitative LC-MS/MS to map the proximity interactome of ATG9A, which included entire protein complexes involved in protein trafficking, and proteins implicated in autophagy but previously lacking any physical link to core autophagy machinery. We also unexpectedly found an ATG9A interaction with an ULK1-independent ATG13-ATG101 dimer that promotes autophagy in fed cells.

Elevated ATG13 in serum of patients with ME/CFS stimulates oxidative stress response in microglial cells via activation of receptor for advanced glycation end products (RAGE).

Myalgic Encephalomyelitis, also known as Chronic Fatigue Syndrome (ME/CFS), is a multisystem illness characterized by extreme muscle fatigue associated with pain, neurocognitive impairment, and chronic inflammation. Despite intense investigation, the molecular mechanism of this disease is still unknown. Here we demonstrate that autophagy-related protein ATG13 is strongly upregulated in the serum of ME/CFS patients, indicative of impairment in the metabolic events of autophagy. A Thioflavin T-based protein aggregation assay, array screening for autophagy-related factors, densitometric analyses, and confirmation with ELISA revealed that the level of ATG13 was strongly elevated in serum samples of ME/CFS patients compared to age-matched controls. Moreover, our microglia-based oxidative stress response experiments indicated that serum samples of ME/CFS patients evoke the production of reactive oxygen species (ROS) and nitric oxide in human HMC3 microglial cells, whereas neutralization of ATG13 strongly diminishes the production of ROS and NO, suggesting that ATG13 plays a role in the observed stress response in microglial cells. Finally, an in vitro ligand binding assay provided evidence that ATG13 employs the Receptor for Advanced Glycation End-products (RAGE) to stimulate ROS in microglial cells. Collectively, our results suggest that an impairment of autophagy following the release of ATG13 into serum could be a pathological signal in ME/CFS.

WGCNA-Based DNA Methylation Profiling Analysis on Allopurinol-Induced Severe Cutaneous Adverse Reactions: A DNA Methylation Signature for Predisposing Drug Hypersensitivity.

BACKGROUND: The role of aberrant DNA methylation in allopurinol-induced severe cutaneous adverse reactions (SCARs) is incompletely understood. To fill the gap, we analyze the DNA methylation profiling in allopurinol-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) patients and identify the DNA methylation signature for predisposing allopurinol hypersensitivity. METHODS: Genome-scale methylation analysis was conducted using the Illumina® HumanMethylation450 BeadChip. Weighted Gene Co-Expression Network Analysis (WGCNA) was utilized to analyze the data. RESULTS: A total of 21,497 annotated promoter regions were analyzed. Ten modules were identified between allopurinol hypersensitivity and tolerance, with turquoise and yellow modules being the most significant correlation. ATG13, EPM2AIP1, and SRSF11 were the top three hub genes in the turquoise module. MIR412, MIR369, and MIR409 were the top three hub genes in the yellow module. Gene Ontology (GO) analysis revealed that the turquoise module was related to the metabolic process in intracellular organelles and the binding of various compounds, proteins, or nucleotides. The yellow module, however, was related to stimulus sensory perception in cytoskeletal elements and the activity of the receptor or transducer. CONCLUSION: DNA methylation plays a vital role in allopurinol-induced SCARs. DNA methylation profiling of SJS/TEN is significantly related to autophagy and microRNAs (miRNAs).

The emerging roles of ATG1/ATG13 kinase complex in plants.

Autophagy is a conserved system from yeast to mammals that mediates the degradation and renovation of cellular components. This process is mainly driven by numerous autophagy-related (ATG) proteins. Among these components, the ATG1/ATG13 complex plays an essential role in initiating autophagy, sensing nutritional status signals, recruiting downstream ATG proteins to the autophagosome formation site, and governing autophagosome formation. In this review, we will focus on the ATG1/ATG13 kinase complex, summarizing and discussing the current views on the composition, structure, function, and regulation of this complex in plants.

Autophagy coordinates chondrocyte development and early joint formation in zebrafish.

Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.