Search PubMed⌕ Search

Biomedical subjects

Noboru Mizushima

Publications and source records attributed to Noboru Mizushima.

49 records · Page 3Linked to original sources
PubMed2004

The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8.

Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 A resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABAA receptor-associated protein (GABARAP), contains a ubiquitin core with two alpha helices, alpha1 and alpha2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among alpha1, alpha2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins.

Amino Acid Sequence↗
PubMed2003

Coronavirus replication complex formation utilizes components of cellular autophagy.

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells. In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

Adenine↗
PubMed2003

In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.

Animals↗
PubMed2003

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.

Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

Alleles↗
PubMed2003

Crystallization and preliminary X-ray analysis of LC3-I.

Aut7/Apg8 is located in the intermediate structures of the autophagosome and plays an essential role in autophagosome formation. The processed form, cleaved at a C-terminus of Gly120 and called LC3-I, was expressed, purified and crystallized in two crystal forms. One form belongs to space group I4(1), with unit-cell parameters a = 84.39, c = 36.89 A. The other form belongs to space group P4(1) or P4(3), with unit-cell parameters a = 60.48, c = 35.28 A. From the latter form, a complete diffraction data set was collected to 2.1 A resolution.

Animals↗
PubMed2003

Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets.

Mammalian enzymes in late cholesterol biosynthesis have been localized uniformly over the endoplasmic reticulum by enzymatic methods. We report here the first mammalian cholesterol biosynthetic enzyme unequivocally localized at the surface of intracellular lipid storage droplets. NAD(P)H steroid dehydrogenase-like protein (Nsdhl), a mammalian C-3 sterol dehydrogenase involved in the conversion of lanosterol into cholesterol, was localized on lipid droplets by immunofluorescence microscopy and subcellular fractionation. Nsdhl was localized on lipid droplets even when cell growth exclusively depended on cholesterol biosynthesis mediated by this enzyme. Depletion of fatty acids in culture medium reduced the development of lipid droplets and caused Nsdhl redistribution to the endoplasmic reticulum. Elevating oleic acid in medium induced well developed, Nsdhl-positive lipid droplets, and simultaneously caused a reduction in cellular conversion of lanosterol into cholesterol. Manipulated human NSDHL with a missense mutation (G205S) causing a human embryonic developmental disorder, congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome, could no longer be localized on lipid droplets. Although the expression of wild-type NSDHL could restore the defective growth of a CHO cholesterol auxotroph, LEX2 in cholesterol-deficient medium, the expression of NSDHL(G205S) failed to do so. These results point to functional significance of the localization of Nsdhl on lipid droplets. Functional significance was also suggested by the colocalization of Nsdhl on lipid droplets with TIP47, a cargo selection protein for mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. These results add to the growing notion that the lipid droplet is an organelle endowed with more complex roles in various biological phenomena.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate.

Macroautophagy is the major intracellular degradation system delivering cytoplasmic components to the lysosome/vacuole. We have shown that, in yeast and mammalian cells, the Apg12-Apg5 protein conjugate, which is formed by a ubiquitin-like system, is essential for autophagosome formation. In yeast, the Apg12-Apg5 conjugate interacts with a small coiled-coil protein, Apg16, to form a approximately 350 kDa multimeric complex. We demonstrate that the mouse Apg12-Apg5 conjugate forms a approximately 800 kDa protein complex containing a novel WD-repeat protein. Because the N-terminal region of this novel protein shows homology with yeast Apg16, we have designated it mouse Apg16-like protein (Apg16L). Apg16L, however, has a large C-terminal domain containing seven WD repeats that is absent from yeast Apg16. Apg16L interacts with both Apg5 and additional Apg16L monomers; neither interaction, however, depends on the WD-repeat domain. In conjunction with Apg12-Apg5, Apg16L associates with the autophagic isolation membrane for the duration of autophagosome formation. Because these features are similar to yeast Apg16, we concluded Apg16L is the functional counterpart of the yeast Apg16. We also found that membrane targeting of Apg16L requires Apg5 but not Apg12. Because WD-repeat proteins provide a platform for protein-protein interactions, the approximately 800 kDa complex is expected to function in autophagosome formation, further interacting with other proteins in mammalian cells.

Amino Acid Sequence↗
PubMed2003

Role of the Apg12 conjugation system in mammalian autophagy.

The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes. It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system. Covalent attachment of Apg12-Apg5 is essential for autophagy. Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses. These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively. Studies using mammalian cells further revealed the function of the Apg12 system. The Apg12-Apg5 conjugate localizes to elongating autophagic isolation membranes. Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome. Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation.

Animals↗
PubMed2002

Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method.

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.

Amino Acid Sequence↗
PubMed2002

Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast.

Autophagy, responsible for the delivery of cytoplasmic components to the lysosome/vacuole for degradation, is the major degradative pathway in eukaryotic cells. This process requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg5. In the yeast Saccharomyces cerevisiae, the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein, Apg16. The Apg12-Apg5 and Apg16 are localized in the cytosol and pre-autophagosomal structures and play an essential role in autophagosome formation. Here we show that the Apg12-Apg5 conjugate and Apg16 form a approximately 350-kDa complex in the cytosol. Because Apg16 was suggested to form a homo-oligomer, we generated an in vivo system that allowed us to control the oligomerization state of Apg16. With this system, we demonstrated that formation of the approximately 350-kDa complex and autophagic activity depended on the oligomerization state of Apg16. These results suggest that the Apg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formation of the approximately 350-kDa complex is required for autophagy in yeast.

Autophagy↗
PubMed2002

SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation.

Mouse SKD1 AAA ATPase is involved in the sorting and transport from endosomes; cells overexpressing a dominant-negative mutant, SKD1(E235Q) were defective in endosomal transport to both the plasma membranes and lysosomes (Yoshimori et al., 2000). In the present study, we demonstrated that overexpression of SKD1(E235Q) using an adenovirus delivery system caused a defect in autophagy-dependent bulk protein degradation. Morphological observations suggested that this inhibition of autophagy results from an impairment of autolysosome formation. SKD1(E235Q) overexpression also inhibited transport from endosomes to autophagosomes, an event normally occurring prior to fusion with lysosomes. These results indicate that SKD1-dependent endosomal membrane trafficking is required for formation of autolysosomes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2002

Autophagosome formation in mammalian cells.

Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles. The molecular mechanism of autophagy has been extensively studied using the yeast, Saccharomyces cerevisiae, during these past 10 years. These studies suggested that the molecular machinery of autophagosome formation is well conserved from yeast to higher eukaryotes. Identification and characterization of the mammalian counterparts of the yeast autophagy proteins has facilitated our understanding of mammalian autophagy, particularly of autophagosome formation. These findings are now being applied to studies on the physiological roles of autophagy in mammals.

Animals↗