Impairment of autophagic degradation of the ubiquitin-and LC3-binding protein "p62" leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser 334 -Ser 344 ) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56 Å resolution revealed interaction of Trp 340 and Leu 343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.Macroautophagy (hereafter referred to as autophagy) is a major pathway for intracellular bulk degradation by the lysosome/vacuole, and its molecular machinery is highly conserved among eukaryotes. In the autophagic process, a small membrane sac (called isolation membrane) elongates to enwrap cytoplasmic materials, including organelles, and subsequently the extended membrane closes to form a double-membrane structure termed autophagosome. The autophagosome fuses with the lysosome/vacuole where the sequestered cytoplasmic contents within the autophagosome are degraded by hydrolases of the lysosome/vacuole (1, 2). This system is required to execute turnover of cytosolic proteins and for removal of unwanted organelles (e.g. called as pexophagy, mitophagy, and reticulophagy).Genetic and molecular studies in the yeast Saccharomyces cerevisiae have identified 18 ATG (autophagy-related genes) essential for autophagosome formation (1). Among them, eight ATG products include two ubiquitin-like conjugation systems essential for autophagy (3, 4). Atg12 is a ubiquitin-like protein covalently linked to Atg5 by catalytic reactions of Atg7 (ubiquitin-activating enzyme) and Atg10 (ubiquitin-conjugating enzyme) (5). Atg12-Atg5 interacts with Atg16, resulting in oligomerization of Atg12-Atg5⅐Atg16 (6). Another ubiquitin-like protein, Atg8 conjugates to a phosphatidylethanolamine (PE). Atg8, synthesized as a precursor form with extra amino acid residues, is processed by Atg4 cysteine protease, which exposes a glycine residue at its C terminus (7). The processed Atg8 is conjugated to PE by Atg7 (ubiquitin-activating enzyme) and Atg3 (ubiquitin-conjugating enzyme) (8). Furthermore, recent studies have revealed that Atg12-Atg5 conjugate functions as a ubiquitin ligase-like enzyme for Atg8 lipidation reaction (9). Finally, the C-terminal glycine of Atg8 covalently conjugates to an amino group of PE (8). Atg8-PE mediates membrane tethering and hemifusion involving the formation of a...

The ubiquitin ligase complex SCF(Fbs1), which contributes to the ubiquitination of glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. In SCF ubiquitin ligases, a diverse array of F-box proteins confers substrate specificity. Fbs1/Fbx2, a member of the F-box protein family, recognizes high-mannose oligosaccharides. To elucidate the structural basis of SCF(Fbs1) function, we determined the crystal structures of the Skp1-Fbs1 complex and the sugar-binding domain (SBD) of the Fbs1-glycoprotein complex. The mechanistic model indicated by the structures appears to be well conserved among the SCF ubiquitin ligases. The structure of the SBD-glycoprotein complex indicates that the SBD primarily recognizes Man(3)GlcNAc(2), thereby explaining the broad activity of the enzyme against various glycoproteins. Comparison of two crystal structures of the Skp1-Fbs1 complex revealed the relative motion of a linker segment between the F-box and the SBD domains, which might underlie the ability of the complex to recognize different acceptor lysine residues for ubiquitination.

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N-Acetylspermine oxidase (APAO) catalyzes the conversion of N-acetylspermine or N-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.