Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.Autophagy is the major lysosome/vacuole-dependent cellular degradative pathway. During autophagy, cytoplasmic constituents including proteins, lipids, and even entire organelles are surrounded by the phagophore, the initial sequestering compartment. The phagophore then expands to form double-membrane vesicles, termed autophagosomes. The completed autophagosomes fuse with lysosomes/vacuoles allowing access of the cargo to the degradative enzymes within this organelle; the resulting breakdown products are released back into the cytosol as building blocks or catabolic substrates (1–3). Autophagy is not only critical for survival during nutrient deprivation but is also involved in various human pathophysiologies, including cancer and neurodegeneration (4).On autophagy induction, AuTophaGy-related (Atg) proteins accumulate at the phagophore assembly site (PAS) and initiate autophagosome formation. Among the 36 known Atg proteins, Atg1 is the only kinase, and it plays a particularly important role in autophagy induction by controlling the movement of other Atg proteins including Atg9 and Atg23 (5) and in the proper organization of the PAS (6, 7). Atg1 interacts with several proteins, including direct binding to Atg13 (which interacts with Atg17) and Atg11; the kinase activity of Atg1 is regulated in part by its binding to, and/or interaction with, some of these components (8, 9).Atg17 constitutively forms a stable protein complex with Atg29 and Atg31 in both growing and nitrogen starvation conditions (10), and Atg31 directly interacts with Atg17 and Atg29 to bridge these two proteins (11). When autophagy is initiated, the Atg17-Atg31-Atg29 complex is first targeted to the PAS and recruits other Atg proteins, including Atg1 and Atg13, highlighting the significance of the ternary complex (12, 13). Along these lines, a single deletion of the ATG17, ATG29, or ATG31 genes results in a dramatic decrease in autophagy activity (14–16).In this study, we examined the role of posttranslational modification of Atg29. We found that Atg29 is a phosphoprotein and that phosphorylation on the C-terminal domain is critical for autophagy activity; the N terminus of Atg29 contains the functional domain, whereas the C terminus plays a regulatory role. We continued and extended our study to include a structural and functional analysis of the Atg17-Atg31-Atg29 complex. Single-particle electron microscopy (EM) reveals that the recombinant Atg17-Atg31-Atg29 complex is present as an elongated S-shaped dimerized structure, with Atg17 forming the backbone. We further demonstrate that Atg29 has the characteristics of an intrinsically disordered protein (IDP), suggesting that the C-terminal half is flexible and capable of altering its conformation on binding to one or more interacting proteins. Finally, we determined that Atg11 is necessary and sufficient to recruit this complex to the PAS and that phosphorylation of Atg29 is required for its interaction with Atg11 and proper PAS localization.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.The balance between overall rates of protein synthesis and degradation determines whether a cell grows or atrophies. It has long been proposed that overall rates of protein synthesis and degradation can be coordinately regulated (1). For example, when hormones (e.g., insulin and insulin-like growth factor-1) and nutrients are plentiful, rates of protein synthesis are high and protein degradation is suppressed, whereas in starving cells, synthesis falls and overall degradation rises. One critical factor coordinating overall synthesis and degradation is the Ser/Thr protein kinase mammalian target of rapamycin (mTOR), which promotes protein translation (2) while suppressing autophagy (lysosomal proteolysis) (3).mTOR’s pleiotropic functions are catalyzed by two distinct kinase complexes: mTORC1, the key component of which is Raptor, and mTORC2, which instead contains Rictor (4, 5). Growth factors act through class I PI3K and Akt kinases to inhibit the tumor suppressor TSC2, and thereby activate mTORC1 (6). Adequate supply of amino acids, especially leucine, can also activate mTORC1, but through a distinct mechanism involving Rag GTPase and lysosomal recruitment of mTORC1 (4). Unlike mTORC1, mTORC2 is insensitive to amino acid supply but is activated by growth factors via mechanisms that remain unclear.Rapamycin is a natural product that selectively inhibits mTORC1 but not mTORC2. Torin1 is a synthetic mTOR inhibitor that blocks ATP-binding to mTOR and thus inactivates both mTORC1 and mTORC2 (7). Both Torin1 and rapamycin inhibit overall protein synthesis, induce autophagosome formation, and thus mimic the effects of starvation. However, Torin1 is much more effective than rapamycin in affecting these two processes because rapamycin inhibits mTORC1 incompletely (7, 8). Because many types of cancer are associated with overactivation of mTOR, rapamycin and other novel mTOR inhibitors are useful in the treatment of certain cancers (9). Rapamycin is widely used in the clinic as an immune suppressor (10). Nevertheless, rapamycin extends lifespan in aged mice, perhaps by triggering similar changes as occur with dietary caloric restriction (11). Additionally, stimulating autophagy by rapamycin can help clear intracellular protein aggregates as they accumulate in many neurodegenerative diseases and reduce their toxicity (12).Eukaryotic cells degrade proteins by both the autophagy-lysosome system and the ubiquitin proteasome system (UPS). Macroautophagy delivers cytoplasmic proteins or organelles into autophagic vacuoles for degradation, and autophagosome formation is rapidly activated by starvation or mTOR inhibition (3). The UPS is responsible for the degradation of most cytosolic and nuclear proteins in mammalian cells, including the short-lived regulatory and misfolded proteins as well as the bulk of cell constituents, which are long-lived components (13, 14). Degradation by the UPS is highly selective, involving the attachment of a ubiquitin (Ub) chain to the substrate through the sequential actions of a Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and one of the cell’s many Ub ligases (E3s) (15). Proteins conjugated to Ub chains are rapidly degraded by the 26S proteasome, whereas the Ub molecules are recycled by proteasome-associated deubiquitinating enzymes (DUBs) (16). Although the degradation of specific short-lived proteins has been studied extensively, our understanding of the global mechanisms controlling proteasomal degradation of the bulk of cell constituents is very limited.The best-characterized function of mTOR is to enhance protein translation through mTORC1-mediated phosphorylation of 4E-BPs and S6Ks (2). The simultaneous suppression of protein degradation is generally attributed to the ability of mTORC1 to inhibit autophagy, which seems to occur by the phosphorylation and inactivation of Atg1/Ulks (3). The goal of this study was to test whether the increased proteolysis upon mTOR inhibition may also occur through activation of the UPS. We demonstrate herein that mTOR inhibition not only enhances lysosomal proteolysis, but also rapidly stimulates the ubiquitination and proteasomal degradation of many proteins. We also investigated the mechanisms by which the UPS is activated, the role of mTORC1 or mTORC2, and the nature of the proteins whose stability is affected by mTOR.Together with the enhancement of autophagy, this activation of proteolysis by the UPS upon mTOR inhibition appears to represent an important adaptation to starvation that helps slow growth and provide essential amino acids. A very different role of mTORC1 in regulating protein degradation was recently proposed by Zhang et al. (17), who surprisingly reported that rapamycin did not rapidly increase proteolysis, but after 16 h actually reduced overall proteolysis by decreasing proteasome expression (17). Such a suppression is inconsistent with the well-established increase in proteolysis and autophagy in starving cells, and various observations about the regulation of the UPS (see below). Zhang et al.’s conclusions probably result from their potentially misleading pulse-chase methods to measure protein degradation and cell culture conditions (18). In this study, we have used well-validated pulse-chase approaches (14) to measure proteolysis by proteasomal and lysosomal systems after mTOR inhibition and have also monitored overall Ub conjugation and proteasome content.     

Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration

Parkinson disease (PD) is a progressive neurodegenerative disorder pathologically characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta and the presence, in affected brain regions, of protein inclusions named Lewy bodies (LBs). The ATP13A2 gene (locus PARK9) encodes the protein ATP13A2, a lysosomal type 5 P-type ATPase that is linked to autosomal recessive familial parkinsonism. The physiological function of ATP13A2, and hence its role in PD, remains to be elucidated. Here, we show that PD-linked mutations in ATP13A2 lead to several lysosomal alterations in ATP13A2 PD patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished lysosomal-mediated clearance of autophagosomes. Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells restores lysosomal function and attenuates cell death. Relevant to PD, ATP13A2 levels are decreased in dopaminergic nigral neurons from patients with PD, in which ATP13A2 mostly accumulates within Lewy bodies. Our results unravel an instrumental role of ATP13A2 deficiency on lysosomal function and cell viability and demonstrate the feasibility and therapeutic potential of modulating ATP13A2 levels in the context of PD.     

SUMOylation at K340 inhibits tau degradation through deregulating its phosphorylation and ubiquitination

Intracellular accumulation of the abnormally modified tau is hallmark pathology of Alzheimer’s disease (AD), but the mechanism leading to tau aggregation is not fully characterized. Here, we studied the effects of tau SUMOylation on its phosphorylation, ubiquitination, and degradation. We show that tau SUMOylation induces tau hyperphosphorylation at multiple AD-associated sites, whereas site-specific mutagenesis of tau at K340R (the SUMOylation site) or simultaneous inhibition of tau SUMOylation by ginkgolic acid abolishes the effect of small ubiquitin-like modifier protein 1 (SUMO-1). Conversely, tau hyperphosphorylation promotes its SUMOylation; the latter in turn inhibits tau degradation with reduction of solubility and ubiquitination of tau proteins. Furthermore, the enhanced SUMO-immunoreactivity, costained with the hyperphosphorylated tau, is detected in cerebral cortex of the AD brains, and β-amyloid exposure of rat primary hippocampal neurons induces a dose-dependent SUMOylation of the hyperphosphorylated tau. Our findings suggest that tau SUMOylation reciprocally stimulates its phosphorylation and inhibits the ubiquitination-mediated tau degradation, which provides a new insight into the AD-like tau accumulation.Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly. Intracellular accumulation of neurofibrillary tangles (NFTs) and extracellular precipitation of senile plaques are the most prominent pathological hallmarks of AD (1–3). The clinical-to-pathological correlation studies have demonstrated that the number of NFTs consisting of hyperphosphorylated tau correlates with the degree of dementia in AD (4–6). Tau is the major microtubule-associated protein that normally contains 2–3 mol of phosphate per mole of tau protein. In AD brains, tau is abnormally hyperphosphorylated (namely AD-P-tau) and the phosphate level increases to 5–9 mol phosphate per mole tau (4). AD-P-tau does not bind to tubulin and become incompetent in promoting microtubule assembly and maintaining the stability of the microtubules. The AD-P-tau also sequesters normal tau from microtubules (7), and serves as a template for the conversion of normal tau into misfolded protein in a prion-like manner (8). In addition to hyperphosphorylation, tau is also contains other posttranslational modifications, such as ubiquitination and SUMOylation (5, 9–11). The abnormal modification of tau also decreases its solubility, and ∼40% of the hyperphosphorylated tau in AD brains has been isolated as sedimentable nonfibril cytosolic protein (1, 12). Although the mechanisms underlying the formation of the NFTs remain unclear, the altered tau modifications and impaired degradation are believed to play a role. Therefore, clarifying the mechanism that may cause tau accumulation is of great significance for understanding the pathogenesis of AD and for developing new therapeutics.Like other proteins, tau can be degraded by autophagy-lysosomal and ubiqutin-proteasomal systems under physiological conditions. In mouse cortical neurons, a C-terminal–truncated form of tau that mimics tau cleaved at Asp421 (tauΔC) is removed by macroautophagic and lysosomal mechanisms (13). Lysosomal perturbation inhibits the clearance of tau with accumulation and aggregation of tau in M1C cells (14). Cathepsin D released from lysosome can degrade tau in cultured hippocampal slices (15). Inhibition of the autophagic vacuole formation leads to a noticeable accumulation of tau (14). Studies also suggest that tau protein is degraded in an ubiquitin-, ATP-, and 26S proteasome-, but not a 20S proteasome-dependent manner under normal conditions (16). When the cells are exposed to the stresses, CHIP, a ubiquitin ligase that interacts directly with Hsp70/90, can induce tau ubiquitination and thus selectively reduce the level of detergent insoluble tau (17). The compensatory activation of autophagy-lysosomal or ubiqutin-proteasomal system can antagonize tau aggregation; therefore, tau accumulation does not show in the early stage of AD. During the evolution of AD, a gradual impairment of autophagy-lysosomal system and ubiqutin-proteasomal system has been detected at later stage of the disease (18–20). Studies suggest that the ubiquitin-mediated degradation pathway seems ineffective in removing the tau-positive fibrillar structures in the AD brains (21–23); however, the mechanisms underlying the impairment of the ubiqutin-proteasomal system are elusive.Ubiquitin is an important component of the cellular defense system that tags abnormal proteins for their degradation by ATP-dependent nonlysosomal proteases (24). Monoclonal antibodies 3-39 and 5-25 raised against paired helical filaments of NFTs have been shown to recognize ubiquitin (25). Meanwhile, tau can be sumoylated at K340 in vitro by SUMO-1 (small ubiquitin-like modifier protein-1) and to a lesser extent by SUMO2 and SUMO3 (9–11). Moreover, SUMO-1 immunoreactivity was colocalized with tau aggregates in neuritic plaques of APP transgenic mice (11). It is well known that SUMO share similarities with ubiquitin in both the structure and the biochemistry of their conjugation (26). Therefore, tau SUMOylation may compete against its ubiquitination and thus suppress tau degradation. In the present study, we found that tau SUMOylation reciprocally stimulates its phosphorylation and thus inhibits the ubiquitination and degradation of tau proteins.     

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [³⁵S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.Phospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slow-twitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca²⁺ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1–3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca²⁺, thereby making SERCA2a less active in transporting Ca²⁺ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca²⁺. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca²⁺/calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca²⁺ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased intracellular Ca²⁺ concentration) stimuli.Previous studies identified three PLN mutations in families of patients with hereditary dilated cardiomyopathy. These mutations, the substitution of Cys for Arg9 (R9C) (4), Arg14 deletion (RΔ14) (5), and the substitution of TGA for TAA in the Leu39 codon, creating a stop codon (L39stop) (6), also lead to dilated cardiomyopathy in transgenic mice. At the cellular level, ectopically expressed RΔ14 and L39stop PLN mutants localize at the plasma membrane in HEK-293T cells, cultured mouse neonatal cardiomyocytes, and cardiac fibroblasts, whereas wild-type and the R9C mutant reside within the endoplasmic reticulum (ER)/SR (6, 7). These data, together with a recent study by Sharma et al. (8), suggest a highly ordered trafficking of PLN, ultimately ensuring correct localization, and thus function, within the SR. However, PLN trafficking and degradation mechanisms in mammalian cardiomyocytes have not been clearly established.Protein degradation and clearance of damaged organelles are critical for cellular physiology, and failure in proper clearance has been shown to have pathological repercussions (9). Autophagy is a major mechanism that mediates protein and organelle degradation in response to external and internal signals. External stimulation through pharmacological agonists, such as metformin and rapamycin, promotes autophagy via AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signal pathways, whereas amino acid starvation and an increased intracellular AMP/ATP ratio serve as internal signals to promote autophagy via the Ca²⁺/Calmodulin-dependent kinase kinase-β (10). Steps in the autophagy pathway involve nucleation of targeted macromolecules on the ER membrane, trafficking of autophagosomes to lysosomes and, finally, fusion of the autophagosome-lysosome, resulting in targeted protein degradation (11). In the heart, autophagy plays a crucial role in response to insults, in part by relieving ER stress (12) and removing damaged mitochondria (13). Loss of autophagy could result in irreversible apoptosis and reduced cardiac functioning (14).To characterize PLN degradation, we conducted a series of assays in cultured mouse neonatal cardiomyocytes (CMNCs) and the hearts of HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (Hace1)-null mice. Our results show that PLN degradation required both polyubiquitinylation and p62-mediated selective autophagy in CMNCs. Loss of HACE1 was associated with increased PLN levels, supporting the notion that selective autophagy modulates PLN degradation in vivo. Metformin promoted wild-type and R9C PLN degradation through autophagic pathways, resulting in metformin-induced inotropic enhancement.     

Membrane recruitment of autophagy proteins in selective autophagy

Autophagy is a stress response that is upregulated in response to signals such as starvation, growth factor deprivation, endoplasmic reticulum stress, and pathogen infection. Defects in this pathway are the underlying cause of a number of diseases, including metabolic aberrations, infectious diseases, and cancer, which are closely related to hepatic disorders. To date, more than 30 human ATG (autophagy) genes have been reported to regulate autophagosome formation. In this review, we summarize the current understanding of how ATG proteins behave during autophagosome formation in both non-selective and selective autophagy.     

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the “safety belt” conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.     

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H₂O₂. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants.Macroautophagy, hereafter referred to as autophagy, is an evolutionarily conserved biological process from yeast to higher eukaryotes including plants (1–4). During autophagy, intracellular components are enclosed in double membrane vesicles called autophagosomes. Cargoes in autophagosomes are then delivered to the vacuole or the lysosome for degradation or recycling. Biogenesis of autophagosomes requires conjugation of phosphatidylethanolamine (PE) to ubiquitin-like Atg8 proteins (1, 5). Before PE conjugation, the Atg8 protein is posttranslationally processed by Atg4 cysteine protease at the conserved C-terminal glycine (Gly) residue (6). The exposed Gly residue is then implicated in adduct with PE. Atg4 is also required for a deconjugation step in which Atg8-PE is removed from the outer membrane of autophagosome for Atg8 recycling and autophagosome completion (7, 8).Compared with single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two AtAtg4 (AtAtg4a and AtAtg4b) and nine AtAtg8 (AtAtg8a–AtAtg8i) genes (9, 10). However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8 isoforms. Because AtAtg4 and AtAtg8 genes are differentially regulated under different environmental conditions, we and others have proposed that the expansion of AtAtg4 and AtAtg8 members may provide specificity for activating autophagy during different biological processes (11–13).To test differential activity of AtAtg4s, we have systematically investigated the specificity of AtAtg4a and AtAtg4b cysteine proteases for processing of different AtAtg8s. For this, we designed unique AtAtg8 synthetic substrates and developed a sensitive method to follow the processing of AtAtg8s. Our results indicate that AtAtg4a is a predominant protease and it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. The AtAtg8 synthetic substrate is also efficiently processed in vivo in Arabidopsis transgenic plants and this processing requires endogenous AtAtg4 proteases. Transgenic Arabidopsis plants expressing the AtAtg8a synthetic substrate described here will be a valuable tool to dissect the autophagy process and its role in different biological processes.     

溶酶体在新城疫病毒诱导肿瘤细胞凋亡和自噬中的作用

溶瘤病毒(oncolytic virus,OV)具有对肿瘤细胞选择性杀伤而对正常细胞无影响的特性,在针对肿瘤治疗方面有着巨大的潜力.凋亡与自噬在溶瘤病毒杀伤肿瘤细胞的过程中扮演者重要的角色,而溶酶体与二者有着密切的联系.新城疫病毒(newcastle disease virus,NDV)在感染肿瘤细胞后,溶酶体不仅会触...     

Fatty acid synthase is preferentially degraded by autophagy upon nitrogen starvation in yeast

Autophagy, an evolutionarily conserved intracellular catabolic process, leads to the degradation of cytosolic proteins and organelles in the vacuole/lysosome. Different forms of selective autophagy have recently been described. Starvation-induced protein degradation, however, is considered to be nonselective. Here we describe a novel interaction between autophagy-related protein 8 (Atg8) and fatty acid synthase (FAS), a pivotal enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids in yeast. We show that although FAS possesses housekeeping functions, under starvation conditions it is delivered to the vacuole for degradation by autophagy in a Vac8- and Atg24-dependent manner. We also provide evidence that FAS degradation is essential for survival under nitrogen deprivation. Our results imply that during nitrogen starvation specific proteins are preferentially recruited into autophagosomesAutophagy is a major cellular catabolic pathway in eukaryotes, responsible for the degradation of organelles and large protein aggregates. This process is induced under various stress conditions, such as amino acid starvation, hypoxia, and oxidative stress (1, 2).In yeast (Saccharomyces cerevisiae), nitrogen starvation is known to induce macroautophagy (hereafter termed autophagy), which is essential mainly for supplementing amino acids needed for protein synthesis (3). Autophagy has long been considered to be a nonselective process except for the recruitment of ribosomes into autophagosomes, which was suggested to be selective under conditions of nitrogen starvation (4). Selective autophagy is thought to play a role mainly in cell homeostasis, and it uses the core autophagy genes and varying sets of proteins for different cargos (5). Autophagy-related protein 8 (Atg8), a key autophagic factor essential for autophagosome biogenesis, also plays a major role in selective autophagy by recruiting cargo proteins into the autophagosome (6).Fatty acid synthase (FAS) is a large (2.6 MDa), essential enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids (7). FAS is composed of two subunits, Fas1 (Fasβ) and Fas2 (Fasα), which are arranged in an α₆β₆ macromolecular complex. Deletion of one of the FAS subunits has been shown to result in degradation of the other (8). Whereas the unassembled Fas2 subunit is short-lived and degrades in the proteasome, the unassembled Fas1, once induced, is degraded by autophagy. Moreover, the FAS complex is known to be delivered to the vacuole under potassium acetate starvation (8).Here we investigated the role of autophagy in the degradation of the FAS complex. We report a physical interaction between Atg8 and FAS, and show that autophagy preferentially degrades FAS and decreases its activity during nitrogen starvation. We suggest that the selective degradation of FAS under nitrogen starvation requires the participation of Atg24 and Vac8, two factors that each play a role in selective autophagy (9–12). Finally, we show that FAS degradation is important for cell viability. We propose that selective degradation of proteins under nitrogen starvation occurs to maintain cell homeostasis.     

Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)

The class III phosphatidylinositol 3-kinase (PI3KC3) is crucial for autophagosome biogenesis. It has been long speculated to nucleate the autophagosome membrane, but the biochemical mechanism of such nucleation activity remains unsolved. We recently identified Barkor/Atg14(L) as the targeting factor for PI3KC3 to autophagosome membrane. Here, we show that we have characterized the region of Barkor/Atg14(L) required for autophagosome targeting and identified the BATS [Barkor/Atg14(L) autophagosome targeting sequence] domain at the carboxyl terminus of Barkor. Bioinformatics and mutagenesis analyses revealed that the BATS domain binds to autophagosome membrane via the hydrophobic surface of an intrinsic amphipathic alpha helix. BATS puncta overlap with Atg16 and LC3, and partially with DFCP1, in a stress-inducible manner. Ectopically expressed BATS accumulates on highly curved tubules that likely represent intermediate autophagic structures. PI3KC3 recruitment and autophagy stimulation by Barkor/Atg14(L) require the BATS domain. Furthermore, our biochemical analyses indicate that the BATS domain directly binds to the membrane, and it favors membrane composed of phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]. By binding preferentially to curved membranes incorporated with PtdIns(3)P but not PtdIns(4,5)P2, the BATS domain is capable of sensing membrane curvature. Thus, we propose a novel model of PI3KC3 autophagosome membrane nucleation in which its autophagosome-specific adaptor, Barkor, accumulates on highly curved PtdIns(3)P enriched autophagic membrane via its BATS domain to sense and maintain membrane curvature.     

KLHL3 regulates paracellular chloride transport in the kidney by ubiquitination of claudin-8

A rare Mendelian syndrome—pseudohypoaldosteronism type II (PHA-II)—features hypertension, hyperkalemia, and metabolic acidosis. Genetic linkage studies and exome sequencing have identified four genes—with no lysine kinase 1 (wnk1), wnk4, Kelch-like 3 (KLHL3), and Cullin 3 (Cul3)—mutations of which all caused PHA-II phenotypes. The previous hypothesis was that the KLHL3–Cul3 ubiquitin complex acted on the wnk4–wnk1 kinase complex to regulate Na⁺/Cl⁻ cotransporter (NCC) mediated salt reabsorption in the distal tubules of the kidney. Here, we report the identification of claudin-8 as a previously unidentified physiologic target for KLHL3 and provide an alternative explanation for the collecting duct’s role in PHA-II. Using a tissue-specific KO approach, we have found that deletion of claudin-8 in the collecting duct of mouse kidney caused hypotension, hypokalemia, and metabolic alkalosis, an exact mirror image of PHA-II. Mechanistically, the phenotypes in claudin-8 KO animals were caused by disruption of the claudin-8 interaction with claudin-4, the paracellular chloride channel, and delocalization of claudin-4 from the tight junction. In mouse collecting duct cells, knockdown of KLHL3 profoundly increased the paracellular chloride permeability. Mechanistically, KLHL3 was directly bound to claudin-8, and this binding led to the ubiquitination and degradation of claudin-8. The dominant PHA-II mutation in KLHL3 impaired claudin-8 binding, ubiquitination, and degradation. These findings have attested to the concept that the paracellular pathway is physiologically regulated through the ubiquitination pathway, and its deregulation may lead to diseases of electrolyte and blood pressure imbalances.Gordon’s syndrome, also known as pseudohypoaldosteronism II (PHA-II) or familial hyperkalemic hypertension, features several metabolic derangements, including hypertension, hyperkalemia, and hyperchloremic metabolic acidosis (1). Mutations in four genes have been found to cause Gordon’s syndrome. Two encode the serine-threonine kinases with no lysine kinases (WNKs) (2). The other two encode proteins important in the cullin-really interesting new gene E3 ubiquitin ligase (CRL) complex—Kelch-like 3 (KLHL3) and Cullin 3 (CUL3) (3, 4). Disease-causing mutations in WNKs are dominant and confer gain of function to augment NaCl reabsorption in the distal convoluted tubule (DCT) by a signaling cascade of SPAK/OSR1 to NCC (5, 6). Mutations in KLHL3 are either recessive or dominant. Recessive mutations include premature termination, frameshift, and splicing alternatives, consistent with loss of function, whereas dominant mutations cluster in the β-propeller domain important in target recognition (3, 4). CUL3 mutations are all dominant and de novo and result in the skipping of exon 9 and in-frame deletion of a 57-aa segment important in maintaining the CRL architecture (3). KLHL3 was found to interact with WNK4 and regulate its ubiquitination and degradation (7, 8). Presumably, loss of KLHL3 function may lead to increases in WNK4 protein levels that, in turn, promote NCC phosphorylation and NaCl reabsorption in the DCT. Compatible with this notion, a knock-in mouse model harboring a dominant mutation (R528H) allele in the KLHL3 gene developed PHA-II–related phenotypes and concomitant increases in WNK4 protein abundance and NCC phosphorylation levels (9).All four PHA-II genes have significant expression in renal tubules, including the DCT, the connecting tubule (CNT), and the collecting duct (CD) (2, 3). Although the PHA-II mechanism in the DCT has been extensively studied, the CNT/CD’s role remains largely elusive. The predominant NaCl transport pathway in the CNT/CD is through epithelial Na⁺ channel (ENaC) mediated Na⁺ reabsorption and electrically coupled paracellular Cl⁻ reabsorption (also known as the chloride shunt) (10, 11). WT WNK4 inhibits ENaC conductivity, whereas PHA-II–causing mutations eliminate WNK4’s inhibition of ENaC, thereby promoting Na⁺ reabsorption in the CNT/CD (12). The disease-causing mutation in WNK4 has also been found to augment paracellular Cl⁻ conductance in renal epithelial cells (13, 14). The hypothesis of unopposed chloride shunt is particularly important to explain hyperchloremia and hyperkalemia in PHA-II. The shunt conductance would favor Cl⁻ reabsorption and decrease the lumen-negative transepithelial voltage as the driving force for K⁺ secretion (15). To reveal the molecular nature of chloride shunt and its potential role in PHA-II pathophysiology, we have generated the KO mouse models for two claudin molecules—claudin-4 and claudin-8—both of which are required to generate paracellular Cl⁻ conductance in vitro (16). The claudin-4 KO mice developed hypotension, hypochloremia, and metabolic alkalosis because of renal loss of Cl⁻ (11). The claudin-8 KO phenocopied claudin-4 KO, which can be mechanistically attributed to their interaction and coassembly into the tight junction (TJ). Most intriguingly, KLHL3 directly interacted with claudin-8 and regulated its ubiquitination and degradation. Loss of KLHL3 in the CD cells increased the TJ conductance to chloride. The dominant PHA-II mutation in KLHL3 abolished its interaction and regulation of claudin-8. These findings have revealed a previously unidentified physiological substrate for KLHL3 and provided important insights of the TJ’s role in PHA-II pathogenesis.     

DDX3-mediated miR-34 expression inhibits autophagy and HBV replication in hepatic cells

HBV entry to the host cells and its successful infection depends on its ability to modulate the host restriction factors. DEAD box RNA helicase, DDX3, is shown to inhibit HBV replication. However, the exact mechanism of inhibition still remains unclear. DDX3 is involved in multitude or RNA metabolism processes including biogenesis of miRNAs. In this study, we sought to determine the mechanism involved in DDX3-mediated HBV inhibition. First, we observed that HBx protein of HBV downregulated DDX3 expression in HBV-infected cells. Overexpression of DDX3 inhibited HBx, HBsAg and total viral load, while its knockdown reversed the result in Hep G2.2.15 cells. Expression of miR-34 was downregulated in HBV-infected cells. Overexpression of pHBV_1.3 further confirmed that HBV downregulates miR-34 expression. Consistent with the previous finding that DDX3 is involved in miRNA biogenesis, we observed that expression of miR-34 positively corelated with DDX3 expression. miRNA target prediction tools showed that miR-34 can target autophagy pathway which is hijacked by HBV for the benefit of its own replication. Indeed, transfection with miR-34 oligos downregulated the expression of autophagy marker proteins in HBV-expressing cells. Overexpression of DDX3 in HBV-expressing cells, downregulated expression of autophagy proteins while silencing of DDX3 reversed the results. These results led us to conclude that DDX3 upregulates miR-34 expression and thus inhibits autophagy in HBV-expressing cells while HBx helps HBV evade DDX3-mediated inhibition by downregulating DDX3 expression in HBV-infected cells.     

TFEB的翻译后修饰对自噬的调节作用

转录因子EB(TFEB)是小眼畸形相关转录因子家族成员,通过调节自噬-溶酶体相关基因的表达在脂质代谢等多种生物过程中发挥重要作用。TFEB的活性与细胞定位可通过蛋白质的翻译后修饰进行调节。本文就TFEB翻译后修饰对自噬的调节作用进行综述,旨在加深对动脉粥样硬化等自噬异常所致疾病发病机制的理解,为相应疾病的防治提供新的思路和干预靶点。     

物理方法构建肝细胞自噬模型

目的应用物理方法构建正常人肝细胞株7702细胞的低氧饥饿自噬模型,为研究细胞自噬对肝脏功能的影响机制奠定基础。方法选用7702细胞,使用完全DMEM培养基,在37℃和5%CO2培养箱中培养(正常对照组),使用Binder三气培养箱,设置温度为37℃,CO2浓度为5%、O2浓度为0.3%来提供缺氧环境,使用无血清DMEM培养细胞来诱导饥饿,将其分为低氧饥饿6、12、18和24 h 4个时间组。用Western Blot检测正常对照组和不同时间点实验组的Beclin-1、Atg5以及LC3蛋白表达水平的变化;通过实时荧光定量PCR法检测各组Beclin-1 mRNA、Atg5 mRNA的表达;转染LC3质粒后利用免疫荧光法观察各组细胞发生自噬的情况。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验;计数资料组间比较采用χ2检验。结果低氧饥饿处理6 h组的Beclin-1、Atg5及LC3的蛋白表达量高于正常对照组及其他处理组;低氧饥饿6 h组较其他各组的Beclin-1mRNA和Atg5 mRNA的表达量显著增高,且其增高最显著,差异均有统计学意义(P值均0.05);低氧饥饿组自噬小体数量均高于正常对照组,其中低氧饥饿6 h组的自噬小体数量最高,差异均有统计学意义(P值均0.05)。结论通过物理方法构建的低氧饥饿可以诱导7702细胞发生自噬,并且在低氧饥饿6 h时,细胞发生自噬最为明显。     

Conserved role for autophagy in Rho1-mediated cortical remodeling and blood cell recruitment

Dynamic regulation of cell shape underlies many developmental and immune functions. Cortical remodeling is achieved under the central control of Rho GTPase pathways that modulate an exquisite balance in the dynamic assembly and disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy), associated with bulk cytoplasmic remodeling through lysosomal degradation, has clearly defined roles in cell survival and death. Moreover, it is becoming apparent that proteins, organelles, and pathogens can be targeted for autophagic clearance by selective mechanisms, although the extent and roles of such degradation are unclear. Here we report a conserved role for autophagy specifically in the cortical remodeling of Drosophila blood cells (hemocytes) and mouse macrophages. Continuous autophagy was required for integrin-mediated hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes disrupted for autophagy were impaired in their recruitment to epidermal wounds. Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating selective autophagy as a novel mechanism for modulating cortical dynamics. These results illuminate a specific and conserved role for autophagy as a regulatory mechanism for cortical remodeling, with implications for immune cell function.     

自噬调控衰老的研究进展

随着机体老化,组织器官可发生不可逆退行性改变,这些变化与疾病和死亡密切相关。自噬是细胞内一种重要的分解代谢过程,在维持细胞稳态和促进长寿中起重要作用,机体衰老后,其自噬调节能力也随之下降。本文综述了自噬与衰老相关疾病的关系,明确自噬调控衰老的相关分子机制可能为治疗衰老相关疾病提供新的靶点。