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Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

ABSTRACT: The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch. Mutagenesis data support the importance of both the interface between ATG12 and ATG5 and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

SUBMITTER: Otomo C

PROVIDER: S-EPMC3540207 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

Otomo Chinatsu C Metlagel Zoltan Z Takaesu Giichi G Otomo Takanori T

Nature structural & molecular biology 20121202 1

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjug ...[more]

PMID: 23202584

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Project description:Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended on Atg12 covalently conjugated to Atg5. In support of this finding, HBV required the E2-like enzymes Atg10 and Atg3, which catalyze or facilitate Atg5-12 conjugation, respectively. Atg10 and Atg3 knockdowns decreased HBV production, while Atg3 overexpression increased virus yields. Mapping analyses demonstrated that the HBV core protein encountered the Atg5-12/16L1 complex via interaction with the intrinsically disordered region of the Atg12 moiety that is dispensable for autophagy function. The role of Atg12 in HBV replication was confirmed by its incorporation into virions. Although the Atg5-12/16L1 complex and Atg3 are essential for LC3 lipidation and, thus, for autophagosome maturation and closure, HBV propagation did not require LC3. Silencing of LC3B, the most abundant LC3 isoform, did not inhibit but rather augmented virus production. Similar augmenting effects were obtained upon overexpression of a dominant negative mutant of Atg4B that blocked the lipid conjugation of the LC3 isoforms and their GABARAP paralogues. Together, our data indicate that HBV subverts early, nondegradative autophagy components as assembly scaffolds, thereby concurrently avoiding autophagosomal destruction.IMPORTANCE Infections with the hepatitis B virus (HBV), an enveloped pararetrovirus, cause about 1 million deaths per year, as current therapies rarely achieve a cure. Understanding the HBV life cycle and concomitant host cell interactions is instrumental to develop new antiviral concepts. Here, we proceeded to dissect the roles of the autophagy machinery in virus propagation. By using RNA interference and overexpression studies in HBV-replicating cell lines, we identified the autophagic Atg5-12/16L1 elongation complex along with Atg10 and Atg3 to be an essential scaffold for HBV nucleocapsid assembly/stability. Deficits in Atg5-12/16L1 and Atg10/Atg3, which normally drive autophagophore membrane expansion, strongly impaired progeny virus yields. HBV gained access to Atg5-12/16L1 via interaction of its core protein with the Atg12 moiety of the complex. In contrast, subsequent autophagosome maturation and closure events were unnecessary for HBV replication, as evidenced by inhibition of Atg8/LC3 conjugation. Interfering with the HBV/Atg12 cross talk may be a tool for virus control.

Project description:BIRC5/Survivin is known as a dual cellular functions protein that directly regulates both apoptosis and mitosis in embryonic cells during embryogenesis and in cancer cells during tumorigenesis and tumor metastasis. However, BIRC5 has seldom been demonstrated as a direct macroautophagy/autophagy regulator in cells. ATG7 expression and ATG12-ATG5-ATG16L1 complex formation are crucial for the phagophore elongation during autophagy in mammalian cells. In this study, we observed that the protein expression levels of BIRC5 and ATG7 were inversely correlated, whereas the expression levels of BIRC5 and SQSTM1/p62 were positively correlated in normal breast tissues and tumor tissues. Mechanistically, we found that BIRC5 negatively modulates the protein stability of ATG7 and physically binds to the ATG12-ATG5 conjugate, preventing the formation of the ATG12-ATG5-ATG16L1 protein complex in human cancer (MDA-MB-231, MCF7, and A549) and mouse embryonic fibroblast (MEF) cells. We also observed a concurrent physical dissociation between BIRC5 and ATG12-ATG5 (but not CASP3/caspase-3) and upregulation of autophagy in MDA-MB-231 and A549 cells under serum-deprived conditions. Importantly, despite the fact that upregulation of autophagy is widely thought to promote DNA repair in cells under genotoxic stress, we found that BIRC5 maintains DNA integrity through autophagy negative-modulations in both human cancer and MEF cells under non-stressed conditions. In conclusion, our study reveals a novel role of BIRC5 in cancer cells as a direct regulator of autophagy. BIRC5 may act as a "bridging molecule", which regulates the interplay between mitosis, apoptosis, and autophagy in embryonic and cancer cells.AbbreviationsACTA1: actin; ATG: autophagy related; BIRC: baculoviral inhibitor of apoptosis repeat-containing; BAF: bafilomycin A1; CQ: chloroquine; CASP3: caspase 3; HSPB1/Hsp27: heat shock protein family B (small) member 1/heat shock protein 27; IAPs: inhibitors of apoptosis proteins; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA.

Dataset Information

Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

Publications

Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

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