Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide-bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62's binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.

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 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.

All 13 lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, were identified in a crystalline bovine heart cytochrome c oxidase (CcO) preparation. The chain lengths and unsaturated bond positions of the fatty acid moieties determined by mass spectrometry suggest that each lipid head group identifies its specific binding site within CcOs. The X-ray structure demonstrates that the flexibility of the fatty acid tails facilitates their effective space-filling functions and that the four phospholipids stabilize the CcO dimer. Binding of dicyclohexylcarbodiimide to the O 2 transfer pathway of CcO causes two palmitate tails of phosphatidylglycerols to block the pathway, suggesting that the palmitates control the O 2 transfer process.The phosphatidylglycerol with vaccenate (cis-D 11 -octadecenoate) was found in CcOs of bovine and Paracoccus denitrificans, the ancestor of mitochondrion, indicating that the vaccenate is conserved in bovine CcO in spite of the abundance of oleate (cis-D 9 -octadecenoate). The X-ray structure indicates that the protein moiety selects cis-vaccenate near the O 2 transfer pathway against trans-vaccenate. These results suggest that vaccenate plays a critical role in the O 2 transfer mechanism.