Showing papers by "Isei Tanida published in 2010"

Measurement of Autophagy in Cells and Tissues

Abstract: Two major proteolysis systems, the ubiquitin-proteasome system, and the autophagy-lysosome system, contribute to degradation of various types of protein and/or protein aggregates. In general, the autophagy-lysosome system is involved in bulk intracellular degradation of proteins and organelles, while the ubiquitin-proteasome system is selective. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes, and LC3-II is degraded by lysosomal hydrolases after the fusion of autophagosomes with lysosomes. Therefore, lysosomal turnover of LC3-II reflects starvation-induced autophagic activity, and detection of LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy. When autophagy is impaired, the level of p62/SQSTM1, a ubiquitin- and LC3-binding protein, is increased in addition to the accumulation of ubiquitinated proteins. Here, we describe basic protocols to analyze endogenous LC3-II, p62, and autophagy-related proteins by immunoblotting, immunofluorescence, and electron microscopy.

Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy

Abstract: Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine. Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-α (IFNα) was evaluated. The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNα enhanced the antiviral effect of IFNα and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNα but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine. The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.