Autophagic Membranes Research Articles

β-Elemene induces apoptosis and autophagy in colorectal cancer cells through regulating the ROS/AMPK/mTOR pathway.

β-Elemene is an effective anti-cancer ingredient extracted from the genus Curcuma (Zingiberaceae familiy). In the present study, we demonstrated that β-elemene inhibited the proliferation of colorectal cancer cells and induced cell cycle arrest in the G2/M phase. In addition, β-elemene induced nuclear chromatin condensation and cell membrane phosphatidylserine eversion, decreased cell mitochondrial membrane potential, and promoted the cleavage of caspase-3, caspase-9 and PARP proteins, indicating apoptosis in colorectal cancer cells. At the same time, β-elemene induced autophagy response, and the treated cells showed autophagic vesicle bilayer membrane structure, which was accompanied by up-regulation of the expression of LC3B and SQSTM1. Furthermore, β-elemene increased ROS levels in colorectal cancer cells, promoted phosphorylation of AMPK protein, and inhibited mTOR protein phosphorylation. In the experiments in vivo, β-elemene inhibited the tumor size and induced apoptosis and autophagy in nude mice. In summary, β-elemene inhibited the occurrence and development of colon cancer xenografts in nude mice, and significantly induced apoptosis and autophagy in colorectal cancer cells in vitro. These effects were associated with regulation of the ROS/AMPK/mTOR signaling. We offered a molecular basis for the development of β-elemene as a promising anti-tumor drug candidate for colorectal cancer.

FYVE2, a phosphatidylinositol 3-phosphate effector, interacts with the COPII machinery to control autophagosome formation in Arabidopsis.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase Secretion-associated Ras-related GTPase 1 (SAR1), which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

Membrane dynamics of ATG4B and LC3 in autophagosome formation

The biogenesis of autophagosomes provides the basis for macroautophagy to capture and degrade intracellular cargoes. Binding of the autophagy-related protein ATG8/LC3 to autophagic membranes is essential to autophagosome formation, which involves the specific and dynamic processing of ATG8/LC3 by cysteine protease ATG4. However, to date, the mechanism whereby ATG4 is recruited to the membranes, the interaction of ATG4 and ATG8/LC3 on the membranes, and its role in the growth of phagophore are not completely understood. Here, we used fluorescence recovery after photobleaching to monitor the turnover of GFP-tagged ATG4B and LC3B in living animal cells. The data show that ATG4B localizes to early autophagic membranes in an LC3B-dependent manner. During autophagy, ATG4B and LC3B undergo rapid cytosol/isolation membrane exchange but not at the cytosol/completed autophagosome. In addition, ATG4B activity controls the efficiency of autophagosome formation by impacting the membrane binding/dissociation of LC3B. These data suggest that ATG4 and LC3 play interdependent roles in the formation of autophagosomes.

A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis.

Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system remodels has been poorly understood. Here we identify a new type of membrane contact formed between the ER–Golgi intermediate compartment (ERGIC) and the ER-exit site (ERES) in the ER–Golgi system, which is essential for promoting autophagosome biogenesis induced by different stress stimuli. The ERGIC–ERES contact is established by the interaction between TMED9 and SEC12 which generates a short distance opposition (as close as 2–5 nm) between the two compartments. The tight membrane contact allows the ERES-located SEC12 to transactivate COPII assembly on the ERGIC. In addition, a portion of SEC12 also relocates to the ERGIC. Through both mechanisms, the ERGIC–ERES contact promotes formation of the ERGIC-derived COPII vesicle, a membrane precursor of the autophagosome. The ERGIC–ERES contact is physically and functionally different from the TFG-mediated ERGIC–ERES adjunction involved in secretory protein transport, and therefore defines a unique endomembrane structure generated upon stress conditions for autophagic membrane formation.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Atg12-Interacting Motif Is Crucial for E2-E3 Interaction in Plant Atg8 System.

Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg8 system and the Atg12 system are essential for autophagy. Atg8 and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively, via enzymatic reactions. The Atg8-PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12-Atg5 conjugate recognizes Atg3, the E2 enzyme for Atg8, and facilitates Atg8-PE conjugation by functioning as the E3 enzyme. Although structural and biochemical analyses have well established the Atg8-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12-ATG3), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12b as a complex with the ATG12b-binding region of ATG3 and revealed that ATG12b recognizes the aspartic acid (Asp)-methionine (Met) motif in ATG3 via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG3, suggesting that the Asp-Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates E2-E3 interaction during Atg8 lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.

Selective increment of phosphatidylserine on the autophagic body membrane in the yeast vacuole.

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

Cornus Officinalis Induction of Pancreatic β-cell Autophagy as Revealed by Increased LC3 Expression and P62 Phosphorylation

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of pancreatic beta cells, diminishing the body’s ability to produce insulin. Currently, over 1.2 million people are affected by this disease in the United States with the only treatment being exogenous insulin. The ideal preventative treatment of diabetes would be to target the interventional window during the critical pre-diabetic stage to inhibit the complete loss of pancreatic β-cells. Cornus officinalis (CO) is considered a source of novel therapeutics for this purpose due to prior reports and work from our laboratory (Mol. and Cell. Endo.2019:494:110491) demonstrating that CO may protect β-cells from autoimmune mediated cytotoxicity and enhance function. To elucidate the molecular mechanism of how CO may be biologically impacting a human pancreatic β-cell line (1.1B4), we previously performed a global and phosphorylation mass spectrometry analysis revealing an increased phosphorylation of p62, which is an important regulator of autophagy. To validate the mass spectrometry results and determine if CO is truly capable of inducing pancreatic β-cell autophagy, we examined LC3 expression which serves as a typical marker for autophagic membranes. 1.1B4 cells were treated with CO for 6 and 12 hours with at concentrations of 500 and 1000 µg/ml, respectively. Cells were fixed with formaldehyde and permeabilized with Triton X-100 followed by fluorescent detection with an LC3 antibody along with DAPI nuclear staining. The cells and nuclei were then imaged using a florescent microscope and it was discovered that upon treatment of CO, an increase in cytoplasmic LC3 puncta was markedly observed as compared to untreated controls. Western analysis also demonstrated increased expression of intracellular LC3 upon CO induction. We then went on to confirm the autophagic process by immunoblotting of p62, which is a cargo receptor for autophagy. Immunoblotting demonstrated increased expression of phosphorylated p62 in a concentration and time dependent manner after treatment with CO. Altogether, CO appears to increase expression in the critical markers of autophagy and suggests that CO may have potential as a T1D interventional therapy by promoting protective autophagy in the pancreatic β-cells.

Selective Autophagy Receptors in Antiviral Defense.

Autophagy ensures the degradation of cytosolic substrates by the lysosomal pathway. Cargoes destined to be eliminated are confined within double-membrane vesicles called autophagosomes, prior to fusion with endolysosomal vacuoles. Autophagy receptors selectively interact with cargoes and route them to elongating autophagic membranes, a process referred to as selective autophagy. Besides contributing to cell homeostasis, selective autophagy constitutes an important cell-autonomous defense mechanism against viruses. We review observations related to selective autophagy receptor engagement during host cell responses to virus infection. We examine the distinct roles of autophagy receptors in antiviral autophagy, consider the strategies viruses have evolved to escape or oppose such restrictions, and delineate the contributions of selective autophagy to the tailoring of antiviral innate responses. Finally, we mention some open and emerging questions in the field.

Deciphering the role of a membrane-targeting domain in assisting endosomal and autophagic membrane localization of a RavZ protein catalytic domain

The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy-related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CAΔα3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs.

TFEB Gene Promoter Variants Effect on Gene Expression in Acute Myocardial Infarction.

Autophagy is involved in many physiological processes. Transcription factor EB (TFEB) is a master regulator of autophagy and coordinates the expression of autophagic proteins, lysosomal hydrolases, and lysosomal membrane proteins. Though autophagy has been implicated in several human diseases, little is known regarding TFEB gene expression and regulation in the process. Since dysfunctional autophagy plays critical roles in acute myocardial infarction (AMI), dysregulated TFEB gene expression may be associated with AMI by regulating autophagy. In this study, the TFEB gene promoter was genetically and functionally analyzed in AMI patients (n = 352) and ethnic-matched controls (n = 337). A total of fifteen regulatory variants of the TFEB gene, including eight single-nucleotide polymorphisms (SNPs), were identified in this population. Among these, six regulatory variants [g.41737274T>C (rs533895008), g.41737144A>G, g.41736987C > T (rs760293138), g.41736806C > T (rs748537297), g.41736635T > C (rs975050638), and g.41736544C > T] were only identified in AMI patients. These regulatory variants significantly altered the transcriptional activity of the TFEB gene promoter. Further electrophoretic mobility shift assay revealed that three of the variants evidently affected the binding of transcription factors. Therefore, this study identified novel TFEB gene regulatory variants which affect the gene expression. These TFEB gene regulatory variants may contribute to AMI development as a rare risk factor.

Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains.

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase that is essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of the two domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to how the C-terminal lipase domain recognizes autophagic bodies to degrade them.

Autophagosomes get wet.

Agudo-Canalejo et al. now report in Nature that the process of wetting, whereby a liquid establishes a contact with a surface, underlies interactions of phase-separated droplets with autophagic membranes.

Autophagosomal Membrane Origin and Formation.

Autophagosome formation is a regulated membrane remodeling process, which involves the generation of autophagosomal membrane precursors (vesicles), the assembly of the autophagosomal membrane precursors to form the phagophore, and phagophore elongation to complete the autophagosome. The sources of the autophagosomal membrane precursors are endomembrane compartments, such as the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), ER-exit sites (ERES), and endosomes. In response to stress, these structures are remodeled, to generate the early autophagosomal membrane precursors. The phagophore assembly site (PAS), which mainly localizes on the ER, harbors the site for autophagosomal membrane assembly, elongation, and completion. ATG proteins, membrane remodeling factors, and autophagic membranes follow a precise choreography to complete the overall process. In this chapter, we briefly discuss our current knowledge on the membrane origins of the autophagosome, as well as autophagosomal precursor generation, assembly, and expansion.

Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2−), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2−. Significant differences in the lipid profiles of control and Atg2− flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.

Fine particulate matter inhibits phagocytosis of macrophages by disturbing autophagy.

Mounting evidence from epidemiological and clinical studies has revealed marked correlations between the air pollutant fine particulate matter (FPM) and respiratory diseases. FPM reaches distal airways and deposits in alveolar regions where it can act directly on alveolar macrophages. However, the detailed effect of FPM on the physiological function of alveolar macrophages and the underlying mechanisms remain unclear. In this study, we showed that exposing THP-1-derived macrophages to FPM led to autophagy dysfunction. FPM activated the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which promoted the expression of autophagy-related 2A (ATG2A) and reactive oxygen species generation. The overexpression of ATG2A enhanced the synthesis of autophagic membranes, and the excessive production of reactive oxygen species caused autophagy flux inhibition through disrupting the lysosomal activity. More importantly, FPM impaired the phagocytic ability of macrophages on Escherichia coli and apoptotic neutrophils. Finally, we showed that restoring autophagy rescued the impairment of phagocytic ability induced by FPM. In summary, these results reveal the molecular mechanism of autophagy dysfunction caused by FPM and provide a novel approach to resolve the impaired function of macrophages in respiratory diseases induced by FPM.

Long-term autophagy is sustained by activation of CCT\u03b23 on lipid droplets

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.

Interplay Between Lipid Metabolism and Autophagy.

Autophagy is a self-eating process of using lysosomes to degrade macromolecular substances (e.g., proteins and organelles) that are damaged, degenerated, or aging. Lipid metabolism is the synthesis and degradation of lipids (e.g., triglycerides, steroids, and phospholipids) to generate energy or produce the structural components of cell membranes. There is a complex interplay between lipid metabolism (e.g., digestion, absorption, catabolism, biosynthesis, and peroxidation) and autophagy machinery, leading to the modulation of cell homeostasis, including cell survival and death. In particular, lipid metabolism is involved in the formation of autophagic membrane structures (e.g., phagophores and autophagosomes) during stress. Moreover, autophagy, especially selective autophagy (e.g., lipophagy, ferritinophagy, clockophagy, and mitophagy), promotes lipid catabolism or lipid peroxidation-induced ferroptosis through the degradation of various substances within the cell. A better understanding of the mechanisms of autophagy and possible links to lipid metabolism will undoubtedly promote potential treatments for a variety of diseases.

Membrane targeting of core autophagy players during autophagosome biogenesis.

Autophagosomes are vital organelles required to facilitate the lysosomal degradation of cytoplasmic cargo, thereby playing an important role in maintaining cellular homeostasis. A number of autophagy-related (ATG) protein complexes are recruited to the site of autophagosome biogenesis where they act to facilitate membrane growth and maturation. Regulated recruitment of ATG complexes to autophagosomal membranes is essential for their autophagic activities and is required to ensure the efficient engulfment of cargo destined for lysosomal degradation. In this review, we discuss our current understanding of the spatiotemporal hierarchy between ATG proteins, examining the mechanisms underlying their recruitment to membranes. A particular focus is placed on the relevance of phosphatidylinositol 3-phosphate and the extent to which the core autophagy players are reliant on this lipid for their localisation to autophagic membranes. In addition, open questions and potential future research directions regarding the membrane recruitment and displacement of ATG proteins are discussed here.

GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes.

SummaryExcessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.