Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Hepatitis C Virus Core Protein Suppresses Mitophagy by Interacting with Parkin in the Context of Mitochondrial Depolarization

2014; Elsevier BV; Volume: 184; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2014.07.024

ISSN

1525-2191

Autores

Yuichi Hara, Izumi Yanatori, Masanori Ikeda, Emi Kiyokage, Sohji Nishina, Yasuyuki Tomiyama, Kazunori Toida, Fumio Kishi, Nobuyuki Kato, Michio Imamura, Kazuaki Chayama, Keisuke Hino,

Tópico(s)

Adenosine and Purinergic Signaling

Resumo

Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1–infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate–induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury. Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1–infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate–induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury. Oxidative stress is present in chronic hepatitis C to a greater degree than in other inflammatory liver diseases.1Farinati F. Cardin R. De Maria N. Della Libera G. Marafin C. Lecis E. Burra P. Floreani A. Cecchetto A. Naccarato R. Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis.J Hepatol. 1995; 22: 449-456Abstract Full Text PDF PubMed Scopus (371) Google Scholar, 2Valgimigli M. Valgimigli L. Trere D. Gaiani S. Pedulli G.F. Gramantieri L. Bolondi L. Oxidative stress EPR measurement in human liver by radical-probe technique: correlation with etiology, histology and cell proliferation.Free Radic Res. 2002; 36: 939-948Crossref PubMed Scopus (97) Google Scholar The hepatitis C virus (HCV) core protein induces the production of reactive oxygen species (ROS)3Okuda M. Li K. Beard M.R. Showalter L.A. Scholle F. Lemon S.M. Weinman S.A. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.Gastroenterology. 2002; 122: 366-375Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 4Moriya K. Nakagawa K. Santa T. Shintani Y. Fujie H. Miyoshi H. Tsutsumi T. Miyazawa T. Ishibashi K. Horie T. Imai K. Todoroki T. Kimura S. Koike K. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar through mitochondrial electron transport inhibition.5Korenaga M. Wang T. Li Y. Showalter L.A. Chan T. Sun J. Weinman S.A. Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.J Biol Chem. 2005; 280: 37481-37488Crossref PubMed Scopus (347) Google Scholar Because the mitochondria are targets for ROS and ROS generators, HCV-induced ROS have the potential to injure mitochondria. In addition, hepatocellular mitochondrial alterations have been observed in patients with chronic hepatitis C.6Barbaro G. Di Lorenzo G. Asti A. Ribersani M. Belloni G. Grisorio B. Filice G. Barbarini G. Hepatocellular mitochondrial alterations in patients with chronic hepatitis C: ultrastructural and biochemical findings.Am J Gastroenterol. 1999; 94: 2198-2205Crossref PubMed Scopus (163) Google Scholar We previously identified a ROS-associated iron metabolic disorder7Nishina S. Hino K. Korenaga M. Vecchi C. Pietrangelo A. Mizukami Y. Furutani T. Sakai A. Okuda M. Hidaka I. Okita K. Sakaida I. Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription.Gastroenterology. 2008; 134: 226-238Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar and demonstrated that transgenic mice expressing the HCV polyprotein develop hepatocarcinogenesis related to mitochondrial injury induced by HCV and iron overload.8Furutani T. Hino K. Okuda M. Gondo T. Nishina S. Kitase A. Korenaga M. Xiao S.Y. Weinman S.A. Lemon S.M. Sakaida I. Okita K. Hepatic iron overload induces hepatocellular carcinoma in transgenic mice expressing the hepatitis C virus polyprotein.Gastroenterology. 2006; 130: 2087-2098Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Therefore, impaired mitochondrial function may play a critical role in the development of hepatocellular carcinoma (HCC) in patients with chronic HCV infection. Conversely, the affected mitochondria are selectively eliminated through the autophagy-dependent degradation of mitochondria (referred to as mitophagy) in both physiological and pathological settings to maintain the mitochondrial quality.9Kim I. Rodriguez-Enriquez S. Lemasters J.J. Selective degradation of mitochondria by mitophagy.Arch Biochem Biophys. 2007; 462: 245-253Crossref PubMed Scopus (1252) Google Scholar, 10Elmore S.P. Qian T. Grissom S. Lemasters J.J. The mitochondrial permeability transition initiates autophagy in rat hepatocytes.FASEB J. 2001; 15: 2286-2287Crossref PubMed Scopus (546) Google Scholar On the basis of these observations, we hypothesized that HCV may suppress mitophagy, which could lead to the sustained presence of affected mitochondria, increased ROS production, and the development of HCC. Mitochondrial membrane depolarization precedes mitophagy induction,11Matsuda N. Sato S. Shiba K. Okatsu K. Saisho K. Gautier C.A. Sou Y.S. Saiki S. Kawajiri S. Sato F. Kimura M. Komatsu M. Hattori N. Tanaka K. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.J Cell Biol. 2010; 189: 211-221Crossref PubMed Scopus (1341) Google Scholar which is selectively controlled by a variety of proteins in mammalian cells, including pentaerythritol tetranitrate–induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin.12Narendra D.P. Jin S.M. Tanaka A. Suen D.F. Gautier C.A. Shen J. Cookson M.R. Youle R.J. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin.PLoS Biol. 2010; 8: e1000298Crossref PubMed Scopus (1992) Google Scholar, 13Geisler S. Holmström K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2030) Google Scholar, 14Vives-Bauza C. Zhou C. Huang Y. Cui M. de Vries R.L. Kim J. May J. Tocilescu M.A. Liu W. Ko H.S. Magrane J. Moore D.J. Dawson V.L. Grailhe R. Dawson T.M. Li C. Tieu K. Przedborski S. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.Proc Natl Acad Sci USA. 2010; 107: 378-383Crossref PubMed Scopus (1234) Google Scholar, 15Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2856) Google Scholar, 16Chan N.C. Salazar A.M. Pham A.H. Sweredoski M.J. Kolawa N.J. Graham R.L. Hess S. Chan D.C. Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.Hum Mol Genet. 2011; 20: 1726-1737Crossref PubMed Scopus (764) Google Scholar, 17Gegg M.E. Cooper J.M. Chau K.Y. Rojo M. Schapira A.H. Taanman J.W. Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy.Hum Mol Genet. 2010; 19: 4861-4870Crossref PubMed Scopus (685) Google Scholar, 18Chen D. Gao F. Li B. Wang H. Xu Y. Zhu C. Wang G. Parkin mono-ubiquitinates Bcl-2 and regulates autophagy.J Biol Chem. 2010; 285: 38214-38223Crossref PubMed Scopus (129) Google Scholar, 19Narendra D. Kane L.A. Hauser D.N. Fearnley I.M. Youle R.J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy: VDAC1 is dispensable for both.Autophagy. 2010; 6: 1090-1106Crossref PubMed Scopus (589) Google Scholar PINK1 facilitates Parkin targeting of the depolarized mitochondria.12Narendra D.P. Jin S.M. Tanaka A. Suen D.F. Gautier C.A. Shen J. Cookson M.R. Youle R.J. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin.PLoS Biol. 2010; 8: e1000298Crossref PubMed Scopus (1992) Google Scholar, 13Geisler S. Holmström K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2030) Google Scholar, 14Vives-Bauza C. Zhou C. Huang Y. Cui M. de Vries R.L. Kim J. May J. Tocilescu M.A. Liu W. Ko H.S. Magrane J. Moore D.J. Dawson V.L. Grailhe R. Dawson T.M. Li C. Tieu K. Przedborski S. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.Proc Natl Acad Sci USA. 2010; 107: 378-383Crossref PubMed Scopus (1234) Google Scholar, 15Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2856) Google Scholar Although Parkin ubiquitinates a broad range of mitochondrial outer membrane proteins,14Vives-Bauza C. Zhou C. Huang Y. Cui M. de Vries R.L. Kim J. May J. Tocilescu M.A. Liu W. Ko H.S. Magrane J. Moore D.J. Dawson V.L. Grailhe R. Dawson T.M. Li C. Tieu K. Przedborski S. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.Proc Natl Acad Sci USA. 2010; 107: 378-383Crossref PubMed Scopus (1234) Google Scholar, 17Gegg M.E. Cooper J.M. Chau K.Y. Rojo M. Schapira A.H. Taanman J.W. Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy.Hum Mol Genet. 2010; 19: 4861-4870Crossref PubMed Scopus (685) Google Scholar, 18Chen D. Gao F. Li B. Wang H. Xu Y. Zhu C. Wang G. Parkin mono-ubiquitinates Bcl-2 and regulates autophagy.J Biol Chem. 2010; 285: 38214-38223Crossref PubMed Scopus (129) Google Scholar, 19Narendra D. Kane L.A. Hauser D.N. Fearnley I.M. Youle R.J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy: VDAC1 is dispensable for both.Autophagy. 2010; 6: 1090-1106Crossref PubMed Scopus (589) Google Scholar it remains unclear how Parkin enables the damaged mitochondria to be recognized by the autophagosome. Structures containing autophagy-related protein 9A and the uncoordinated family member-51–like kinase 1 complex independently target depolarized mitochondria at the initial stages of Parkin-mediated mitophagy, whereas the autophagosomal microtubule-associated protein light chain 3 (LC3) is critical for efficient incorporation of damaged mitochondria into the autophagosome at a later stage.20Itakura E. Kishi-Itakura C. Koyama-Honda I. Mizushima N. Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy.J Cell Sci. 2012; 125: 1488-1499Crossref PubMed Scopus (213) Google Scholar LC3-I undergoes post-translational modification by phosphatidylethanolamine to become LC3-II, and LC3-II insertion into the autophagosomal membrane is a key step in autophagosome formation. In addition, the autophagic adaptor p62 is recruited to mitochondrial clusters and is essential for mitochondrial clearance,13Geisler S. Holmström K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2030) Google Scholar although it remains controversial as to whether p62 is essential for mitochondrial recognition by the autophagosome13Geisler S. Holmström K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2030) Google Scholar or rather is important for perinuclear clustering of depolarized mitochondria.19Narendra D. Kane L.A. Hauser D.N. Fearnley I.M. Youle R.J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy: VDAC1 is dispensable for both.Autophagy. 2010; 6: 1090-1106Crossref PubMed Scopus (589) Google Scholar, 21Okatsu K. Saisho K. Shimanuki M. Nakada K. Shitara H. Sou Y.S. Kimura M. Sato S. Hattori N. Komatsu M. Tanaka K. Matsuda N. p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria.Genes Cells. 2010; 15: 887-900PubMed Google Scholar Our aim was to examine whether HCV suppresses mitophagy. We found that HCV core protein inhibits the translocation of Parkin to affected mitochondria by interacting with Parkin and subsequently suppressing mitophagy. These results imply that mitochondria affected by HCV core protein are unlikely to be eliminated, which may intensify oxidative stress and increase the risk of hepatocarcinogenesis. HCV-Japanese fulminant hepatitis-1 (JFH1)–infected Huh7 cells have previously been described in detail.22Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Kräusslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2415) Google Scholar The supernatants were collected from cell culture–generated JFH1-Huh7 cells at 21 days after infection and stored until use at −80°C after filtering through a 0.45-μm filter. For infection experiments with the HCV-JFH1 virus, 1 × 105 Huh7 cells per well were plated onto 6-well plates and cultured for 24 hours. Then, we infected the cells with 50 μL (equivalent to a multiplicity of infection of 0.1) of inoculum. The culture supernatants were collected, and the levels of the HCV core were determined using an enzyme-linked immunosorbent assay (ELISA; Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan). Total RNA was isolated from the infected cellular lysates using an RNeasy mini kit (Qiagen, Hilden, Germany) for quantitative RT-PCR analysis of the intracellular HCV RNA. The HCV infectivity in the culture supernatants was determined by a focus-forming assay at 48 hours after infection. The HCV-infected cells were detected using an anti-HCV core antibody (CP-9 and CP-11, Institute of Immunology, Ltd, Tokyo, Japan). Intracellular HCV infectivity was determined using a focus-forming assay at 48 hours after inoculation of the lysates by repeated freeze-and-thaw cycles (three times). To depolarize the mitochondria, the cells were treated with 10 μmol/L carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma-Aldrich, St. Louis, MO) for 1 to 2 hours or 1 μmol/L valinomycin (Sigma-Aldrich) for 3 hours; CCCP represses ATP synthesis through the loss of the H+ gradient without affecting mitochondrial electron transport, which is known to induce mitochondrial fragmentation.13Geisler S. Holmström K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2030) Google Scholar The pAlbSVPA-HCV vector, which contains the full-length polyprotein-coding region under the control of the murine albumin promoter/enhancer, has previously been described in detail.23Li K. Prow T. Lemon S.M. Beard M.R. Cellular response to conditional expression of hepatitis C virus core protein in Huh7 cultured human hepatoma cells.Hepatology. 2002; 35: 1237-1246Crossref PubMed Scopus (91) Google Scholar, 24Lerat H. Honda M. Beard M.R. Loesch K. Sun J. Yang Y. Okuda M. Gosert R. Xiao S.Y. Weinman S.A. Lemon S.M. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar Of the four transgenic lineages with evidence of RNA transcription of the full-length HCV-N open reading frame (FL-N), the FL-N/35 lineage proved capable of breeding large numbers.24Lerat H. Honda M. Beard M.R. Loesch K. Sun J. Yang Y. Okuda M. Gosert R. Xiao S.Y. Weinman S.A. Lemon S.M. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar Urokinase-type plasminogen activator–transgenic severe combined immunodeficiency mice were generated, and human hepatocytes were transplanted to generate chimeric mice.25Tateno C. Yoshizane Y. Saito N. Kataoka M. Utoh R. Yamasaki C. Tachibana A. Soeno Y. Asahina K. Hino H. Asahara T. Yokoi T. Furukawa T. Yoshizato K. Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am J Pathol. 2004; 165: 901-912Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar The chimeric mice were injected with genotype 1b HCV-positive human serum samples, as described previously.26Kimura T. Imamura M. Hiraga N. Hatakeyama T. Miki D. Noguchi C. Mori N. Tsuge M. Takahashi S. Fujimoto Y. Iwao E. Ochi H. Abe H. Maekawa T. Arataki K. Tateno C. Yoshizato K. Wakita T. Okamoto T. Matsuura Y. Chayama K. Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice.J Gen Virol. 2008; 89: 2108-2113Crossref PubMed Scopus (32) Google Scholar The mouse livers were extracted 12 weeks after the infection, when the serum HCV RNA titers had increased over baseline levels. Male FL-N/35 transgenic mice, age-matched C57BL/6 mice (control), and chimeric mice with and without HCV infection were fed, maintained, and then euthanized by i.p. injection of 10% nembutal sodium, according to the guidelines approved by the Institutional Animal Care and Use Committee. The study protocol for obtaining human serum samples conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Committee. HCV RNA26Kimura T. Imamura M. Hiraga N. Hatakeyama T. Miki D. Noguchi C. Mori N. Tsuge M. Takahashi S. Fujimoto Y. Iwao E. Ochi H. Abe H. Maekawa T. Arataki K. Tateno C. Yoshizato K. Wakita T. Okamoto T. Matsuura Y. Chayama K. Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice.J Gen Virol. 2008; 89: 2108-2113Crossref PubMed Scopus (32) Google Scholar and human albumin25Tateno C. Yoshizane Y. Saito N. Kataoka M. Utoh R. Yamasaki C. Tachibana A. Soeno Y. Asahina K. Hino H. Asahara T. Yokoi T. Furukawa T. Yoshizato K. Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am J Pathol. 2004; 165: 901-912Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar were quantified as described previously. Human albumin levels in the serum of chimeric mice were determined using the Human Albumin ELISA Quantification kit (Bethyl Laboratories Inc., Montgomery, TX). The mitochondrial membrane potential (ΔΨ) was measured using a Cell Meter JC-10 Mitochondrial Membrane Potential Assay kit (AAT Bioquest, Inc., Sunnyvale, CA), according to the manufacturer's instructions. The fluorescent intensities for both J-aggregates (red) and monometric forms (green) of JC-10 were measured at Ex/Em = 490/525 nm and 540/590 nm with a Varioskan Flush Multimode Reader (Thermo Fisher Scientific, Waltham, MA). The cells were lysed by mechanical homogenization using a small pestle, and mitochondrial extraction was performed using a Qproteome Mitochondria Isolation kit (Qiagen), according to the manufacturer's instructions. Liver mitochondria were isolated as described previously with some modifications.27Ando M. Korenaga M. Hino K. Ikeda M. Kato N. Nishina S. Hidaka I. Sakaida I. Mitochondrial electron transport inhibition in full genomic hepatitis C replicon cells is restored by reducing viral replication.Liver Int. 2008; 28: 1158-1166Crossref PubMed Scopus (21) Google Scholar In brief, the livers were minced on ice and homogenized by five strokes with a Dounce homogenizer and a tight-fitting pestle in isolation buffer [70 mmol/L sucrose, 1 mmol/L KH2PO4, 5 mmol/L HEPES, 220 mmol/L mannitol, 5 mmol/L sodium succinate, and 0.1% bovine serum albumin (BSA), pH 7.4]. The homogenate was centrifuged at 800 × g for 5 minutes at 4°C. The supernatant fraction was retained, whereas the pellet was washed with isolation buffer and centrifuged again. The combined supernatant fractions were centrifuged at 1000 × g for 15 minutes at 4°C to obtain a crude mitochondrial pellet. The cellular ROS level was measured by oxidation of the cell-permeable, oxidation-sensitive fluorogenic precursor, 2′,7′-dihydrodichlorofluorescein diacetate (Molecular Probes Inc., Eugene, OR). Fluorescence was measured using a Varioskan Flush Multimode Reader at 495/535 nm (excitation/emission). Mitochondrial pellets were measured for total glutathione [reduced glutathione (GSH) + oxidized glutathione (GSSG)] and GSH content using the GSSG/GSH Quantification kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The concentration of GSH was calculated using the following formula:GSHconcentration=Totalglutathioneconcentration−[GSSGconcentration]×2(1) The liver tissue samples (approximately 50 mg) were minced in ice-cold metaphosphoric acid solution, homogenized, and centrifuged at 3000 × g for 10 minutes at 4°C. Lysates from the liver tissue samples and mitochondrial samples (2 mg) were evaluated for the concentration of GSH using the thioester method and a GSH-400 kit (Oxis International Inc., Portland, OR) and for total glutathione content using the glutathione reductase–dinitrothiocyanobenzene recycling assay and the GSH-412 kit (Oxis International Inc.), as described previously.5Korenaga M. Wang T. Li Y. Showalter L.A. Chan T. Sun J. Weinman S.A. Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.J Biol Chem. 2005; 280: 37481-37488Crossref PubMed Scopus (347) Google Scholar Samples were lysed in radioimmunoprecipitation assay buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 50 mmol/L NaF, 1 mmol/L Na3VO4, 0.1% SDS, and 0.5% Triton X-100], as described previously,28Kim S.J. Syed G.H. Siddiqui A. Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy.PLoS Pathog. 2013; 9: e1003285Crossref PubMed Scopus (138) Google Scholar supplemented with 1% protease inhibitor mixture (Sigma-Aldrich) and 100 mmol/L phenylmethylsulfonyl fluoride. Cell lysates or mitochondrial pellets were subjected to immunoblot analysis using an iBlot Gel Transfer Device (Invitrogen, Carlsbad, CA). The membranes were incubated with the following primary antibodies: rabbit anti-human LC3 (Novus Biologicals, Littleton, CO), rabbit anti-human p62/SQSTM1 (MBL, Nagoya, Japan), rabbit anti-human Parkin (Cell Signaling Technology, Danvers, MA), mouse anti-human Parkin (Santa Cruz Biotechnology, Inc.), rabbit anti-human p-Parkin (Ser 378; Santa Cruz Biotechnology, Inc.), rabbit anti-human PINK1 (Cell Signaling Technology), mouse anti-human mitochondrial heat shock protein-70 (BioReagents, Golden, CO), mouse anti-human ubiquitin (Santa Cruz Biotechnology, Inc.), goat anti-human voltage-dependent anion-selective channel protein 1 (VDAC1; Santa Cruz Biotechnology, Inc.), monoclonal antisynthetic HCV core peptide (CP11; Institute of Immunology, Ltd), mouse anti-HCV non-structural (NS) 3 protein (Abcam, Cambridge, MA), mouse anti-HCV NS4A (Abcam), mouse anti-HCV NS5A protein (Abcam), and rabbit anti-human β-actin (Cell Signaling Technology). To address the detail localization of core and Parkin, the cells treated with CCCP for 1 hour were fixed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 mol/L Millonig's phosphate buffer (pH 7.4) for 30 minutes. The cells were incubated with a mixture of the following primary antibodies in phosphate-buffered saline (PBS) containing 1% BSA and 0.05% sodium azide overnight at 20°C: mouse monoclonal antisynthetic HCV core peptide (Institute of Immunology), rabbit anti-human Parkin (Abcam), and rabbit anti-rat LC3 (Wako Pure Chemical Industries, Ltd, Osaka, Japan). After washing with PBS, the cells were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., Baltimore Pike, PA) in 1% BSA for 2 hours at 20°C. After washing with PBS, the cells were incubated with Alexa Fluor-488 FluoroNanogold-streptavidin (Jackson ImmunoResearch Laboratories, Inc.), indocarbocyanine-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.), and indocarbocyanine-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) in 1% BSA for 2 hours at 20°C. After washing with PBS, the cells were incubated with mouse peroxidase–anti-peroxidase complex (Jackson ImmunoResearch Laboratories, Inc.) in PBS for 3 hours at 20°C. The peroxidase reduction was developed with 0.05% diaminobenzidine tetrahydrochloride in 50 mmol/L Tris buffer containing 0.01% hydrogen peroxide for 20 minutes at room temperature. The diameter of the gold immunoparticles was increased using a silver enhancement kit (HQ silver; Nanoprobes, Inc., Yaphank, NY) for 4 minutes at room temperature. After treatment with 1% osmium and 2% uranyl acetate, the cells were dehydrated in a graded series of ethanol and embedded in Epon-Araldite (OKEN, Tokyo, Japan). Serial ultrathin sections (each 70 nm thick) were examined using an electron microscope (model JEM1400; JEOL, Tokyo, Japan). These immune–electron microscopic methods were generally performed according to our previous study.29Toida K. Kosaka K. Aika Y. Kosaka T. Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb, IV: intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons.Neuroscience. 2000; 101: 11-17Crossref PubMed Scopus (90) Google Scholar The cells were fixed, permeabilized, and immunostained with rabbit anti-human Parkin (Abcam), goat anti-human Parkin (Santa Cruz Biotechnology, Inc.), goat anti-human Tom20 (Santa Cruz Biotechnology, Inc.), rabbit anti-rat LC3 (Wako Pure Chemical Industries, Ltd), or mouse monoclonal antisynthetic HCV core peptide (Institute of Immunology) antibodies, followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.), fluorescein isothiocyanate–conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc.), or Alexa Fluor 647–conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). Cell images were captured using a confocal microscope (model LSM700; Zeiss, Jena, Germany) equipped with 488-, 555-, and 639-nm diodes. The images were acquired in a sequential mode using a 63× Plan Apochromat numerical aperture/1.4 oil objective and the appropriate filter combinations. All images were saved as tagged image file format files. The contrast was adjusted using Photoshop version CS5 (Adobe, San Jose, CA), and the images were imported into Illustrator version CS5 (Adobe). Colocalization was assessed with line scans using ImageJ software version 1.46 (NIH, Bethesda, MD). Coimmunoprecipitation was performed using a Dynabeads Co-Immunoprecipitation Kit (Invitrogen), according to the manufacturer's instructions. Magnetic beads (Dynabeads M-270 Epoxy) were conjugated to anti-VDAC1 (Santa Cruz Biotechnology, Inc.), anti-Parkin (Cell Signaling Technology), anti-ubiquitin (Santa Cruz Biotechnology, Inc.), or anti-p62 (MBL) antibodies by rotating overnight at 37°C. The antibody-Dynabeads complex was then treated with coupling buffer. Beads coupled to anti-VDAC, anti-Parkin, anti-ubiquitin, or anti-p62 were incubated with cell lysates for 30 minutes at 4°C and then washed with coupling buffer. Collected protein complexes were subjected to immunoblot analysis using anti-VDAC, anti-ubiquitin (Santa Cruz Biotechnology, Inc.), and

Referência(s)