Mammalian autophagy degrades nuclear constituents in response to tumorigenic stress

During autophagy, double-membrane autophagosomes are observed in the cytoplasm. Thus, extensive studies have focused on autophagic turnover of cytoplasmic material. Whether autophagy has a role in degrading nuclear constituents is poorly understood. We reveal that the autophagy protein LC3/Atg8 directly interacts with the nuclear lamina protein LMNB1 (lamin B1), and binds to LMN/lamin-associated chromatin domains (LADs). Through these interactions, autophagy specifically mediates destruction of nuclear lamina during tumorigenic stress, such as by activated oncogenes and DNA damage. This nuclear lamina degradation upon aberrant cellular stress impairs cell proliferation by inducing cellular senescence, a stable form of cell-cycle arrest and a tumor-suppressive mechanism. Our findings thus suggest that, in response to cancer-promoting stress, autophagy degrades nuclear material to drive cellular senescence, as a means to restrain tumorigenesis. Our work provokes a new direction in studying the role of autophagy in the nucleus and in tumor suppression.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.     

NBR1-dependent selective autophagy is required for efficient cell-matrix adhesion site disassembly

Macroautophagy/autophagy has classically been recognized for its vital role in supporting cellular survival during various stresses. However, emerging work has demonstrated that selective autophagy has an impact on diverse cell biological processes by mediating the degradation of various cellular contents during normal cellular homeostasis. We recently established that selective autophagy supports cell migration by promoting the turnover of integrin-based cell-matrix adhesion sites, or focal adhesions (FAs). The autophagy cargo receptor NBR1 acts as a critical mediator of this pathway by promoting targeting of autophagosomes to FAs, leading to their disassembly via the sequestration of FA proteins. Our results demonstrate FAs as a new cellular target for selective autophagy.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Substrate recognition in selective autophagy and the ubiquitin–proteasome system

Dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation and adaptation. Eukaryotic cells utilize two mechanistically distinct but largely complementary systems — the 26S proteasome and the lysosome (or vacuole in yeast and plants) — to effectively target a wide range of proteins for degradation. The concerted action of the ubiquitination machinery and the 26S proteasome ensures the targeted and tightly regulated degradation of a subset of commonly short-lived cellular proteins. Autophagy is a distinct degradation pathway, which transports a highly heterogeneous set of cargos in dedicated vesicles, called autophagosomes, to the lysosome. There the cargo becomes degraded and its molecular building blocks are recycled. While general autophagy randomly engulfs portions of the cytosol, selective autophagy employs dedicated cargo adaptors to specifically enrich the forming autophagosomes for a certain type of cargo as a response to various intra- or extracellular signals. Selective autophagy targets a wide range of cargos including long-lived proteins and protein complexes, organelles, protein aggregates and even intracellular microbes. In this review we summarize available data on cargo recognition mechanisms operating in selective autophagy and the ubiquitin–proteasome system (UPS), and emphasize their differences and common themes. Moreover, we derive general regulatory principles underlying cargo recognition in selective autophagy, and describe the system-wide crosstalk between these two cellular protein degradation systems. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407–4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434–1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.     

Role of ATG8 and autophagy in programmed nuclear degradation in Tetrahymena thermophila

Autophagy is an evolutionarily conserved mechanism for the degradation of cellular components, but its role in enucleation during differentiation has not been established. Tetrahymena thermophila is a unicellular eukaryote with two functionally distinct nuclei, the somatic (macro-) and the germ line (micro-) nuclei. These nuclei are produced during sexual reproduction (conjugation), which involves differentiation and selective degradation of several specific nuclei. To examine the role of autophagy in nuclear degradation, we studied the function of two ATG8 genes in Tetrahymena. Through fluorescent protein tagging, we found that both proteins are targeted to degrading nuclei at specific stages, with some enrichment on the nuclear periphery, suggesting the formation of autophagosomes surrounding these nuclei. In addition, ATG8 knockout mutant cells showed a pronounced delay in nuclear degradation without apparently preventing the completion of other developmental events. This evidence provided direct support for a critical role for autophagy in programmed nuclear degradation. The results also showed differential roles for two ATG8 genes, with ATG8-65 playing a more significant role in starvation than ATG8-2, although both are important in nuclear degradation.     

Don't forget to be picky – selective autophagy of protein aggregates in neurodegenerative diseases

The homeostasis of cells depends on the selective degradation of damaged or superfluous cellular components. Autophagy is the major pathway that recognizes such components, sequesters them in de novo formed autophagosomes and delivers them to lysosomes for degradation. The recognition of specific cargo and the biogenesis of autophagosomes involve a dedicated machinery of autophagy related (ATG) proteins. Intense research over the past decades has revealed insights into the function of autophagy proteins and mechanisms that govern cargo recognition. Other aspects including the molecular mechanisms involved in the onset of human diseases are less well understood. However, autophagic dysfunctions, caused by age related decline in autophagy or mutations in ATG proteins, are directly related to a large number of human pathologies including neurodegenerative disorders. Here, we review most recent discoveries and breakthroughs in selective autophagy and its relationship to neurodegeneration.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H 2O 2) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.     

Finding a ribophagy receptor

Although ribophagy was demonstrated in budding yeast a decade ago, a specific receptor for this process has been unknown. Recently, a study revealed that NUFIP1 (nuclear FMR1 interacting protein 1) functions as a receptor for the selective degradation of ribosomes by starvation-induced autophagy in cultured mammalian cells. In addition to the identification of a selective autophagy receptor, this study suggests a strategy that can be adapted to the identification of additional novel receptor proteins.     

The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells

The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes‐ and obesity‐regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re‐localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi‐associated ATPase enhancer of 16 kDa (GATE16) and microtubule‐associated protein 1A/1B‐light chain 3. Gain‐of‐function and loss‐of‐function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H₂O₂) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP-LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.