Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable approximately 3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1-Atg13-FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7 À/À mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7 À/À liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cellintrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51–like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.

Objectives Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of rare autoimmune diseases in which both genetic and environmental factors play important roles. To identify genetic factors of IIM including polymyositis (PM), dermatomyositis (DM), and clinically amyopathic DM (CADM), we performed the first genome-wide association study (GWAS) for IIM in an Asian population.Methods We genotyped and tested 496,819 SNPs for association using 576 IIM patients and 6,270 control subjects. We also examined the causal mechanism of disease associated variants by in silico analyses using publicly available data sets as well as by in in vitro analyses using reporter assays and apoptosis assays. ResultsWe identified a variant in WDFY4 that was significantly associated with CADM (rs7919656; odds ratio (OR)=3.87; P=1.5×10 Furthermore, we demonstrated that both WDFY4 and tr-WDFY4 interacted with pattern recognition receptors such as TLR3, TLR4, TLR9, and MDA5 and augmented the NF-κB activation by these receptors. WDFY4 isoforms also enhanced MDA5-induced apoptosis, to a greater extent in the tr-WDFY4-transfected cells.Conclusions As CADM is characterized by the appearance of anti-MDA5 autoantibodies and severe lung inflammation, the WDFY4 variant may play a critical role in the pathogenesis of CADM.

ObjectivesThe hallmark histopathology of PM is the presence of CD8+ T cells in the non-necrotic muscle cells. The aim of this study was to clarify the pathological significance of CD8+ T cells in muscle cells.MethodsC2C12 cells were transduced retrovirally with the genes encoding MHC class I (H2Kb) and SIINFEKL peptide derived from ovalbumin (OVA), and then differentiated to myotubes (H2KbOVA-myotubes). H2KbOVA-myotubes were co-cultured with OT-I CD8+ T cells derived from OVA-specific class I restricted T cell receptor transgenic mice as an in vitro model of PM to examine whether the CD8+ T cells invade into the myotubes and if the myotubes with the invasion are more prone to die than those without. Muscle biopsy samples from patients with PM were examined for the presence of CD8+ T cells in muscle cells. The clinical profiles were compared between the patients with and without CD8+ T cells in muscle cells.ResultsAnalysis of the in vitro model of PM with confocal microscopy demonstrated the invasion of OT-I CD8+ T cells into H2KbOVA-myotubes. Transmission electron microscopic analysis revealed an electron-lucent area between the invaded CD8+ T cell and the cytoplasm of H2KbOVA-myotubes. The myotubes invaded with OT-I CD8+ T cells died earlier than the uninvaded myotubes. The level of serum creatinine kinase was higher in patients with CD8+ T cells in muscle cells than those without these cells.ConclusionCD8+ T cells invade into muscle cells and contribute to muscle injury in PM. Our in vitro model of PM is useful to examine the mechanisms underlying muscle injury induced by CD8+ T cells.