Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease.

A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice.

It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice.

Metabolic contribution of hepatic autophagic proteolysis: old wine in new bottles.

Pioneering work on autophagy was achieved soon after the discovery of lysosomes more than 50 years ago. Due to its prominent lysosomal activity and technical ease of handling, the liver has been at the center of continuous and vigorous investigations into autophagy. Many important discoveries, including suppression by insulin and plasma amino acids and stimulation by glucagon, have been made through in vivo and in vitro studies using perfused liver and cultured hepatocytes. The long-term controversy about the origin and nature of the autophagosome membrane has finally led to the conclusion of "phagophore," through extensive molecular cell biological approaches enlightened by the discovery of autophagy-essential ATG genes. Furthermore, recent studies using liver-specific autophagy-deficient mice have thrown light on the unique role of a selective substrate of autophagy, p62. The stabilized p62 accumulating in autophagy-deficient liver manipulates Nrf2-dependent transcription activation through specific binding to Keap1, which results in the elevated gene expression involved in detoxification. This is the first example of the dysregulation of gene expression under autophagy deficiency. Thus, basal liver autophagy makes a large contribution to the maintenance of cell homeostasis and health. Meanwhile, precise comparisons of wild-type and liver-specific autophagy-deficient mice under starvation conditions have revealed that amino acids released by autophagic degradation can be metabolized to produce glucose via gluconeogenesis for the maintenance of blood glucose, and can also be excreted to the circulation to supply serum amino acids. These results strongly confirm that induced liver autophagy plays a pivotal role in metabolic compensation. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Differences in expression patterns of cathepsin C/dipeptidyl peptidase I in normal, pathological and aged mouse central nervous system.

Cathepsin C (CC) (EC 3.4.14.1, dipeptidyl peptidase I) is a lysosomal cysteine protease that is required for the activation of several granule-associated serine proteases in vivo. CC has been shown to be constitutively expressed in various tissues, but the enzyme is hardly detectable in central nervous system (CNS) tissues. In the present study, we investigated the regional and cellular distribution of CC in normal, aging and pathological mouse brains. Immunoblotting failed to detect CC protein in whole brain tissues of normal mice, as previously described. However, low proteolytic activity of CC was detected in a brain region-dependent manner, and granular immunohistochemical signals were found in neuronal perikarya of particular brain regions, including the accessory olfactory bulb, the septum, CA2 of the hippocampus, a part of the cerebral cortex, the medial geniculate, and the inferior colliculus. In aged mice, the number of CC-positive neurons increased to some extent. The protein level of CC and its proteolytic activity showed significant increases in particular brain regions of mouse models with pathological conditions--the thalamus in cathepsin D-deficient mice, the hippocampus of ipsilateral brain hemispheres after hypoxic-ischemic brain injury, and peri-damaged portions of brains after penetrating injury. In such pathological conditions, the majority of the cells that were strongly immunopositive for CC were activated microglia. These lines of evidence suggest that CC is involved in normal neuronal function in certain brain regions, and also participates in inflammatory processes accompanying pathogenesis in the CNS.

Ferulic acid induces mammalian target of rapamycin inactivation in cultured mammalian cells.

Ferulic acid (FA), a naturally occurring polyphenol abundant in vegetables and rice bran, is known to possess a potent antioxidant activity, thereby protecting cells from oxidative stress. In the present study, we show that in addition to its known anti-oxidant activity, ferulic acid exerts substantial inhibitory activity on cellular mammalian target of rapamycin (mTor)-signaling pathways. In HeLa cells and mouse primary hepatocytes cultured with conventional nutrient-rich media, ferulic acid (1 mM) elicited dephosphorylation of S6 kinase and its substrate ribosomal S6. The dephosphorylating activity of ferulic acid was almost comparable to that of rapamycin, an established mTor inhibitor (TORC1). We next investigated the effect of ferulic acid on autophagy, a major cellular degradative process, which significantly contributes to the maintenance of cell homeostasis. Using a conventional green fluorescent protein-microtubule-associated protein IA/IB light chain 3 (GFP-LC3) dot assay to evaluate autophagy flux, we showed that ferulic acid caused a significant increase in GFP-LC3 dots under serum-rich conditions in HeLa cells. The enhancement of autophagic flux by ferulic acid was almost equivalent to that of rapamycin. Furthermore, ferulic acid significantly enhanced autophagic degradation of (14)C-leucine-labeled long-lived proteins of cultured mouse hepatocytes under nutrient-rich conditions, but not nutrient-deprived conditions. These results indicate that ferulic acid is almost the equivalent of rapamycin in the ability to inhibit mTor (TORC1), which makes it a potent activator of basal autophagy.

Crucial role for autophagy in degranulation of mast cells.

BACKGROUND: Autophagy plays a crucial role in controlling various biological responses including starvation, homeostatic turnover of long-lived proteins, and invasion of bacteria. However, a role for autophagy in development and/or function of mast cells is unknown. OBJECTIVE: To investigate a role for autophagy in mast cells, we generated bone marrow-derived mast cells (BMMCs) from mice lacking autophagy related gene (Atg) 7, an essential enzyme for autophagy induction. METHODS: Bone marrow-derived mast cells were generated from bone marrow cells of control and IFN-inducible Atg7-deficient mice, and morphologic and functional analyses were performed. RESULTS: We found that conversion of type I to type II light chain (LC3)-II, a hallmark of autophagy, was constitutively induced in mast cells under full nutrient conditions, and LC3-II localized in secretory granules of mast cells. Although deletion of Atg7 did not impair the development of BMMCs, Atg7(-/-) BMMCs showed severe impairment of degranulation, but not cytokine production on FcεRI cross-linking. Intriguingly, LC3-II but not LC3-I was co-localized with CD63, a secretory lysosomal marker, and was released extracellularly along with degranulation in Atg7(+/+) but not Atg7(-/-) BMMCs. Moreover, passive cutaneous anaphylaxis reactions were severely impaired in mast cell-deficient WBB6F1-W/W(V) mice reconstituted with Atg7(-/-) BMMCs compared with Atg7(+/+) BMMCs. CONCLUSION: These results suggest that autophagy is not essential for the development but plays a crucial role in degranulation of mast cells. Thus, autophagy might be a potential target to treat allergic diseases in which mast cells are critically involved.

Peroxisome degradation in mammals.

This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated.

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells.

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of approximately 31 and approximately 60kDa in cell lysates, and the higher expression of approximately 31kDa band was further increased after stimulation with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS). A approximately 31kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.

Comprehensive proteomics analysis of autophagy-deficient mouse liver.

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.

Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination.

BACKGROUND/AIM: Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required. MATERIALS AND METHODS: A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis. RESULTS: The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement. CONCLUSION: These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor (EGFR) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B (FAM83B) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR. Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR. Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR.

A novel gene expression scoring system for accurate diagnosis of basaloid squamous cell carcinoma of the esophagus.

Basaloid squamous cell carcinoma of the esophagus (BSCE) is a rare variant of squamous cell carcinoma that is difficult to distinguish from other carcinomas by preoperative endoscopic biopsy because of its histological varieties. Accurate diagnosis is essential for adequate treatment, and the methods proposed so far (e.g., immunohistochemical staining) have limitations. In this study, we tried to identify the characteristic bundles of gene expression in BSCE using comprehensive gene expression analysis (CGEA). Subsequently, we constructed a gene expression scoring system for the proper diagnosis of BSCE. Fifty-seven surgical specimens, including seven BSCEs, obtained from 30 patients who underwent esophagectomy were used for constructing the scoring system. Three hundred and twelve biopsy specimens, including eight BSCEs, obtained from 80 patients and 20 commercially available formalin-fixed paraffin-embedded (FFPE) specimens diagnosed as esophageal cancer, including 13 BSCEs, were used for validation. After our original mathematical extraction algorithm, 75 genes were extracted to distinguish BSCE from non-BSCE. The cumulative converted values (gene expression score) of the respective 75 genes from each specimen were obtained and lined up in ascending order to assess the optimal gene expression cut-off score for a definitive diagnosis of BSCE. The validation of this scoring system showed high prediction of the biopsy specimens [area under the curve (AUC)=0.981; 95% confidence interval (CI): 0.952­1.000] and the commercially available FFPE specimens (AUC=0.901; 95% CI: 0.750-1.000). In conclusion, using CGEA in a gene expression scoring system helps in differentiating BSCE from non-BSCE with high accuracy and may contribute in improving BSCE treatment.

FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma.

Personalized therapy for non­small cell lung cancer (NSCLC), particularly lung adenocarcinoma, has recently been significantly improved by the discovery of various molecular targets. However, this has not been the case for lung squamous cell carcinoma (SCC). In the present study, we identified the family with sequence similarity 83, member B (FAM83B) as a candidate marker for SCC through a comprehensive gene expression analysis and examined its correlations with various clinicopathological factors. The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection from 2005 to 2011 at the Fukushima Medical University Hospital (Fukushima, Japan). They included 102 patients with adenocarcinoma and 113 with SCC. FAM83B expression was first examined in some of the samples by gene expression analysis and western blotting, and then all clinical specimens were evaluated by immunohistochemistry (IHC). The relationship between the quantitative values for IHC and clinicopathological factors was statistically analyzed. The results showed that FAM83B mRNA expression was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this trend. Specimens containing >10% positive area for FAM83B were judged as 'positive'; 94.3% (107/113) of SCC and 14.7% (15/102) of adenocarcinoma were positive. Patients were divided into two subgroups according to expression (54 high­expression and 53 low­expression patients); the high­expression group was associated with a better disease­free survival (DFS) rate (P=0.042, log­rank test). In conclusion, FAM83B may be a reliable diagnostic and prognostic biomarker for SCC. Detailed analyses of FAM83B function in lung cancer are required to understand how its expression is associated with better prognosis in SCC.

The intracellular location and function of proteins of neuronal ceroid lipofuscinoses.

Neuronal ceroid lipofuscinoses are a group of diseases characterized by accumulation of hydrophobic proteins in lysosomes of neurons and other types of cells. NCLs are caused by at least 8 mutant genes (CLN1-CLN8), though CLN4 and CLN7 have not yet been identified. Except for Cln1p, the protein encoded by CLN1, the defective proteins are associated with lysosomal accumulation of mitochondrial ATP synthase subunit c. Cln1p and Cln2p are soluble lysosomal enzymes, targeted to lysosomes in a mannose 6-phosphate dependent manner. Mutations in the lysosomal protease cathepsin D cause another NCL. Cln3p, Cln5p, Cln6p and Cln8p are thought to be transmembrane proteins. Cln3p and Cln5p are localized in the endosome-lysosomal compartment. Deficiency of endosomal membrane protein CLC-3, a member of the chloride channel family, causes NCL-like phenotype and lysosomal storage of subunit c. Herein, we review the features of NCL and NCL-related proteins and discuss the involvement of the proteins in lysosomal degradation of subunit c.

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation.

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin-proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.

Interleukin-11 links oxidative stress and compensatory proliferation.

Apoptotic cells can stimulate the compensatory proliferation of surrounding cells to maintain tissue homeostasis. Although oxidative stress is associated with apoptosis and necrosis, whether it contributes to compensatory proliferation is unknown. Here, we showed that interleukin-11 (IL-11), a member of the IL-6 family of proinflammatory cytokines, was produced by cells in an oxidative stress-dependent manner. IL-11 production depended on the activation in dying cells of extracellular signal-regulated kinase 2, which in turn caused the phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 was subsequently recruited to the Il11 promoter and activated gene transcription. Upon acute liver injury in mice, IL-11 was mainly produced by hepatocytes in response to reactive oxygen species that were presumably released from dying hepatocytes. IL-11 that was secreted by the dying cells then induced the phosphorylation of the transcription factor STAT3 in adjacent healthy hepatocytes, which resulted in their compensatory proliferation. Furthermore, an IL-11 receptor (IL-11R) agonist enhanced the proliferation of hepatocytes and ameliorated oxidative stress upon acetaminophen-induced liver injury. Conversely, the effects of acetaminophen were exacerbated in mice deficient in the IL-11R α subunit. Together, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.

Liver autophagy contributes to the maintenance of blood glucose and amino acid levels.

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 understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.

Method for monitoring pexophagy in mammalian cells.

The abundance of peroxisomes within a cell is rapidly controlled depending on environmental changes and physiological conditions. It is well established that phthalate esters can cause a marked proliferation of peroxisomes (Yokota, 1986). Following induction of peroxisomes by a 2-week treatment with phthalate esters in mouse livers, peroxisomal degradation via autophagy can be induced for the subsequent week after discontinuation of the phthalate esters. Autophagic degradation of peroxisomes can be monitored by electron microscopy as well as biochemical assay for some peroxisome markers. Although most of the excess peroxisomes in the liver are selectively degraded within one week, this rapid removal is exclusively impaired in the autophagy-deficient liver.

Structural basis for sorting mechanism of p62 in selective autophagy.

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 (Ser334-Ser344) 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 angstroms resolution revealed interaction of Trp340 and Leu343 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.