CALCOCO1 is a soluble reticulophagy receptor

ABSTRACT

The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells and plays critical roles in diverse processes in metabolism, signaling and intracellular organization. In response to stress stimuli such as nutrient deprivation, accumulation of misfolded proteins or exposure to chemicals, the ER increases in size through upregulated synthesis of its components to counteract the stress. To restore physiological size, the excess ER components are continuously dismantled and degraded by reticulophagy, a form of autophagy that targets, via adaptor molecules called reticulophagy receptors, specific ER portions to the lysosome for degradation. Previous studies have identified several ER resident proteins as reticulophagy receptors. In a recent study, we identified CALCOCO1 as a soluble reticulophagy receptor for the degradation of tubular ER in response to proteotoxic and starvation-induced stress. On the ER membrane, CALCOCO1 interacts with VAPA and VAPB via a FFAT-like motif and recruits autophagy machinery by binding directly to Atg8-family proteins via LIR and UDS interacting region (UIR) motifs acting co-dependently. Depletion of CALCOCO1 in cultured cells led to an impaired ER degradation during stress.     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).     

Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat

The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as “ER stress”. Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS

Abstract

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.     

Selective endosomal microautophagy is starvation-inducible in Drosophila

Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging-like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, whereas selective degradation of single proteins has only been described for chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI). These 2 autophagic pathways are specific for proteins containing KFERQ-related targeting motifs. Using a KFERQ-tagged fluorescent biosensor, we have identified an eMI-like pathway in Drosophila melanogaster. We show that this biosensor localizes to late endosomes and lysosomes upon prolonged starvation in a KFERQ- and Hsc70-4- dependent manner. Furthermore, fly eMI requires endosomal multivesicular body formation mediated by ESCRT complex components. Importantly, induction of Drosophila eMI requires longer starvation than the induction of MA and is independent of the critical MA genes atg5, atg7, and atg12. Furthermore, inhibition of Tor signaling induces eMI in flies under nutrient rich conditions, and, as eMI in Drosophila also requires atg1 and atg13, our data suggest that these genes may have a novel, additional role in regulating eMI in flies. Overall, our data provide the first evidence for a novel, starvation-inducible, catabolic process resembling endosomal microautophagy in the Drosophila fat body.     

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells

Autophagy is one of the cellular adaptive processes that provide protection against many pathological conditions like infection, cancer, neurodegeneration, and aging. Recent evidences suggest that ubiquitination plays an important role in degradation of proteins or defective organelle either through proteasome or autophagy. In this study, we describe the role of TRIM13, ER resident ubiquitin E3 ligase in induction of autophagy and its role during ER stress. The ectopic expression of TRIM13 in HEK-293 cells induces autophagy. Domain mapping showed that coiled-coil (CC) domain is required for induction of autophagy. TRIM13 is stabilized during ER stress, interacts with p62/SQSTM1 and co-localizes with DFCP1. TRIM13 regulates initiation of autophagy during ER stress and decreases the clonogenic ability of the cells. This study for the first time demonstrates the role of TRIM13 in induction of autophagy which may play an important role in regulation of ER stress and may act as tumor suppressor.     

COPII mitigates ER stress by promoting formation of ER whorls

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.Subject terms: Endoplasmic reticulum, Collective cell migration     

Mechanistic insights in recov-ER-phagy: micro-ER-phagy to recover from stress

ABSTRACT

Physiological and pathological stresses may cause swelling of the endoplasmic reticulum (ER), a biosynthetic organelle in eukaryotic cells. Upon conclusion of the stress, ER size and content return to physiological levels. The translocon component SEC62 decorates the portions of excess ER that must be cleared from cells. Our recent paper highlights the role of endosomal sorting complex required for transport (ESCRT)-III-driven micro-ER-phagy in ER remodeling during cell recovery from ER stress.     

内质网应激诱导自噬的分子机制

在真核细胞中,内质网对蛋白质的折叠和运输至关重要,多种病理因素对内质网稳态的扰乱,可导致内质网腔中未折叠或错误折叠蛋白蓄积,即内质网应激(ERS)。细胞为此通过激活一种叫做未折叠蛋白反应(UPR)的防御反应来恢复内质网稳态。自噬是一种被描述为"自我吞食"的细胞代谢过程,其通过批量清除和降解未折叠蛋白以及破损细胞器在ERS时作为一种重要的保护机制。近年的研究显示这两个系统动态互联,且ERS可以通过多种方式诱导自噬的发生。在本文中,我们将总结目前关于ERS尤其是UPR诱导自噬的分子机制的相关知识,以进一步指导关于ERS与自噬关系的的研究。     

Ready player one? Autophagy shapes resistance to photodynamic therapy in cancers

Photodynamic therapy (PDT) is a procedure used in cancer therapy that has been shown to be useful for certain indications. Considerable evidence suggests that PDT might be superior to conventional modalities for some indications. In this report, we examine the relationship between PDT responsiveness and autophagy, which can exert a cytoprotective effect. Autophagy is an essential physiological process that maintains cellular homeostasis by degrading dysfunctional or impaired cellular components and organelles via a lysosome-based pathway. Autophagy, which includes macroautophagy and microautophagy, can be a factor that decreases or abolishes responses to various therapeutic protocols. We systematically discuss the mechanisms underlying cell-fate decisions elicited by PDT; analyse the principles of PDT-induced autophagy, macroautophagy and microautophagy; and present evidence to support the notion that autophagy is a critical mechanism in resistance to PDT. A combined strategy involving autophagy inhibitors may be able to further enhance PDT efficacy. Finally, we provide suggestions for future studies, note where our understanding of the relevant molecular regulators is deficient, and discuss the correlations among PDT-induced resistance and autophagy, especially microautophagy.     

Selective and Non-Selective Autophagic Degradation of Mitochondria in Yeast

Mitochondria are essential to oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondrial homeostasis also plays a crucial role in ageing and programmed cell death, and recent data have suggested that mitochondria degradation is a strictly regulated process. Autophagy is an evolutionary conserved mechanism that provides cells with a mechanism for the continuous turnover of damaged and obsolete macromolecules and organelles. In this work, we investigated mitochondria degradation by autophagy. Electron microscopy observations of yeast cells submitted to nitrogen starvation after growth on different carbon sources provided evidence that microautophagy, rather than macroautophagy, preferentially occurred in cells grown under non-fermentable conditions. The observation of mitochondria degradation showed that both a selective process and a non-selective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.     

Localized de novo phospholipid synthesis drives autophagosome biogenesis

ABSTRACT

During (macro)autophagy, cells form transient organelles, termed autophagosomes, to target a broad spectrum of substrates for degradation critical to cellular and organismal health. Driven by rapid membrane assembly, an initially small vesicle (phagophore) elongates into a large cup-shaped structure to engulf substrates within a few minutes in a double-membrane autophagosome. In particular, how autophagic membranes expand has been a longstanding question. Here, we summarize our recent work that delineates a pathway that drives phagophore expansion by localized de novo phospholipid synthesis. Specifically, we found that the conserved acyl-CoA synthetase Faa1 localizes to nucleated phagophores to locally activate fatty acids for de novo phospholipid synthesis in the neighboring ER. These newly synthesized phospholipids are then preferentially incorporated into autophagic membranes and drive the expansion of the phagophore into a functional autophagosome. In summary, our work uncovers molecular principles of how cells coordinate phospholipid synthesis and flux with autophagic membrane formation during autophagy.

Abbreviations: ACS: acyl-CoA synthestases; CoA: coenzyme A; ER: endoplasmic reticulum     

A life-span extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for life-span extension, it is controversial which forms or aspects of autophagy are responsible for this effect. We addressed this topic by analyzing the life span of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macroautophagy and several forms of microautophagy were dispensable for life-span extension. The only autophagy protein that is critical for life-span extension was Atg15, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for life-span extension, which was indicated by the shortened life span of mutants missing proteins (ypt7Delta, nyv1Delta, vac8Delta) or lipids (erg6Delta) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Delta accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macroautophagy is not blocked by erg6Delta, we propose that a new form of autophagy transports Atg15 via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in life-span extension.     

Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

ObjectivesTargeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.Materials and MethodsThe effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca²⁺ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting.ResultsPR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca²⁺ and then stimulated autophagy through Ca²⁺‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619.ConclusionsOur findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.     

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase–3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase–3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H₂O₂ and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.     

Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

内质网在纳米材料毒性效应形成中的作用及机制

随着纳米材料在食品、药物、生物医学等多领域的应用,其在生产使用过程中对人类健康的影响引起了广泛关注.内质网是蛋白质折叠与加工修饰、脂质合成以及Ca~(2+)储存的主要场所,是维护细胞内稳态的重要细胞器.内质网作为纳米材料的主要靶细胞器之一,在纳米材料引起的毒性效应中起重要作用.本文结合近年来国内外相关研究进展,阐述了纳米银(Ag-NPs)、纳米金(Au-NPs)、纳米二氧化钛(TiO_2-NPs)、纳米氧化锌(ZnO-NPs)、纳米二氧化硅(SiO_2-NPs)、富勒烯(C_(60))、单壁与多壁碳纳米管(SWCNTs/MWCNTs)以及石墨烯与氧化石墨烯(GO)等典型纳米材料对内质网结构与功能的影响,并归纳总结了内质网在不同纳米材料诱导的毒性效应中的作用及其异同点.纳米材料可通过引起内质网应激诱导细胞凋亡、炎症反应以及细胞自噬,还可通过激活IP_3信号通路诱导内质网Ca~(2+)释放激活钙依赖的细胞凋亡.纳米材料可在内质网中积累造成结构损伤及功能障碍,还可诱导内质网自噬.     

A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast S. cerevisiae

BackgroundMacroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions.ObjectiveIn search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast S. cerevisiae that could measure both selective and general autophagy in real time.MethodsFirefly and Renilla luciferase reporter genes were cloned under POT-1 promoter. Using fatty acid medium the promoter was induced and the luciferase cargo was allowed to build up. The cells were then transferred to starvation conditions to stimulate autophagy and the degradation of luciferase markers was followed with time.Results and conclusionThe assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.