The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.     

Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29

Atg17, in complex with Atg29 and Atg31, constitutes a key module of the Atg1 kinase signaling complex and functions as an important organizer of the phagophore assembly site in the yeast Saccharomyces cerevisiae. We have determined the three-dimensional reconstruction of the full S. cerevisiae Atg17-Atg31-Atg29 complex by single-particle electron microscopy. Our structure shows that Atg17-Atg31-Atg29 is dimeric and adopts a relatively rigid and extended “S-shape” architecture with an end-to-end distance of approximately 345 Å. Subunit mapping analysis indicated that Atg17 mediates dimerization and generates a central rod-like scaffold, while Atg31 and Atg29 form two globular domains that are tethered to the concave sides of the scaffold at the terminal regions. Finally, our observation that Atg17 adopts multiple conformations in the absence of Atg31 and Atg29 suggests that the two smaller components play key roles in defining and maintaining the distinct curvature of the ternary complex.     

Structure of Atg5.Atg16, a complex essential for autophagy

Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5.Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97A resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.     

Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.     

Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.     

Structural insights into Atg10-mediated formation of the autophagy-essential Atg12-Atg5 conjugate

The Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 of?KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme.     

A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate

Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns[3]P) to promote autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate, and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1-deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5, and PtdIns(3)P provide the fusion specificity between autophagosomes and lysosomes and that the assembly of this complex initiates the autophagosome maturation process.     

Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.     

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.     

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an ~800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Three isoforms of the Atg16L1 protein contribute different autophagic properties

The mammalian Atg16L1 protein consists of a coiled-coil domain and a tryptophan-aspartic acid (WD) repeat domain and is involved in the process of autophagy. However, the mechanisms underlying the effect of the Atg16L1 isoforms on autophagy remain to be elucidated in humans. In the present study, we successfully cloned three isoforms: Atg16L1-1, which contains the complete sequence; Atg16L1-2, which lacks all of exon 8; and Atg16L1-3, which lacks the coiled-coil domain. Subsequent experiments showed that the three isoforms of Atg16L1 were colocalised with MDC within the cells. Quantitative analysis of fluorescence showed that the average number of dots of Atg16L1-1 that colocalised with MDC was higher than those of Atg16L1-2 and Atg16L1-3. The three isoforms of Atg16L1 also colocalised with the lysosome within the cells. The average number of dots of Atg16L1-1 that colocalised with the lysosome was higher than those of Atg16L1-2 and Atg16L1-3. However, although Atg16L1-1 and Atg16L1-3 colocalised with the mitochondria, Atg16L1-2 did not. Functional analysis showed that overexpression of the three isoforms of Atg16L1 had a stimulative effect on autophagy. Significant increase in the number of positive LC3-II dots per cell was observed in Atg16L1-1 (70.2 ± 2.39 dots); this number was greater than those of the other two isoforms. Atg16L1-2 appeared to have an average of 59.25 ± 2.22 LC3-II dots per cell. Atg16L1-3 appeared to have the least number of LC3-II dots per cell (48.25 ± 2.22 dots) (P < 0.001). Our results indicated that the degree of autophagy varied with different Atg16L1 isoforms. The different domains of Atg16L1 played different roles in the process of autophagy. The coiled-coil domain of Atg16L1 was involved in the process of autophagy.     

Rab33B and its autophagic Atg5/12/16L1 effector assist in hepatitis B virus naked capsid formation and release

Hepatitis B virus morphogenesis is accompanied by the production and release of non‐enveloped capsids/nucleocapsids. Capsid particles are formed inside the cell cytosol by multimerization of core protein subunits and ultimately exported in an uncommon coatless state. Here, we investigated potential roles of Rab GTPases in capsid formation and trafficking by using RNA interference and overexpression studies. Naked capsid release does not require functions of the endosome‐associated Rab5, Rab7 and Rab27 proteins, but depends on functional Rab33B, a GTPase participating in autophagosome formation via interaction with the Atg5‐Atg12/Atg16L1 complex. During capsid formation, Rab33B acts in conjunction with its effector, as silencing of Atg5, Atg12 and Atg16L1 also impaired capsid egress. Analysis of capsid maturation steps revealed that Rab33B and Atg5/12/16L1 are required for proper particle assembly and/or stability. In support, the capsid protein was found to interact with Atg5 and colocalize with Atg5/12/16L1, implicating that autophagy pathway functions are involved in capsid biogenesis. However, a complete and functional autophagy pathway is dispensable for capsid release, as judged by knockdown analysis of Atg8/LC3 family members and pharmaceutical ablation of canonical autophagy. Experiments aimed at analysing the capsid release‐stimulating activity of the Alix protein provide further evidence for a link between capsid formation and autophagy.     

牛γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。     

An Atg13 protein-mediated self-association of the Atg1 protein kinase is important for the induction of autophagy

Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.