Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12–Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.

Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O⨪₂ gateway in Mn-SOD.

P62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly under...