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• W2018082393 abstract "Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5−/− mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5−/− MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation. Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5−/− mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5−/− MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation. Chronic neurodegenerative disorders, such as Alzheimer and Parkinson diseases as well as amyotrophic lateral sclerosis, are a heterogeneous group of diseases characterized by selective loss of neurons in specific regions of the CNS. Despite their heterogeneity, they have similar features, including abnormal deposition of ubiquitinated protein aggregates in inclusion bodies within neurons in the respective affected areas of the CNS (reviewed in Ref. 1Mayer R.J. Drug News Perspect. 2003; 16: 103-108Crossref PubMed Scopus (41) Google Scholar). The ubiquitinated protein aggregates are thought to result from dysfunction of the ubiquitin/proteasome pathway (UPP) 2The abbreviations used are: UPPubiquitin/proteasome pathwayCQchloroquineEpxepoxomicinLC3light chain 33-MA3-methyladenineMEFmouse embryonic fibroblastPLAproximity ligation assayRptregulatory particle triple-A ATPaseSucsuccinylAMC7-amino-4-methylcoumarinCMAchaperone-mediated autophagy. or from structural changes in the protein substrates that prevent their degradation by the UPP (reviewed in Ref. 2Ciechanover A. Cell Death Differ. 2005; 12: 1178-1190Crossref PubMed Scopus (273) Google Scholar). Emerging studies also implicate autophagy impairment in the formation of the ubiquitinated protein aggregates. Accordingly, two recent studies described prominent ubiquitin-positive aggregates in neurons of autophagy-deficient (Atg5−/− or Atg7−/−) mice (3Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Nature. 2006; 441: 885-889Crossref PubMed Scopus (3162) Google Scholar, 4Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Nature. 2006; 441: 880-884Crossref PubMed Scopus (2874) Google Scholar). Based on these results and on the finding that there was no apparent alteration in proteasome activity in the brains of autophagy-deficient mice (4Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Nature. 2006; 441: 880-884Crossref PubMed Scopus (2874) Google Scholar), it was suggested that autophagy acts continuously to dispose of diffuse ubiquitinated proteins in a housekeeping role (3Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Nature. 2006; 441: 885-889Crossref PubMed Scopus (3162) Google Scholar, 4Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Nature. 2006; 441: 880-884Crossref PubMed Scopus (2874) Google Scholar, 5Klionsky D.J. Nature. 2006; 441: 819-820Crossref PubMed Scopus (44) Google Scholar). ubiquitin/proteasome pathway chloroquine epoxomicin light chain 3 3-methyladenine mouse embryonic fibroblast proximity ligation assay regulatory particle triple-A ATPase succinyl 7-amino-4-methylcoumarin chaperone-mediated autophagy. Although the UPP and autophagy were thought to work in parallel, recent investigations suggest a functional link between the two proteolytic pathways (reviewed in Ref. 6Koga H. Kaushik S. Cuervo A.M. Ageing Res. Rev. 2011; 10: 205-215Crossref PubMed Scopus (318) Google Scholar). The sequestosome 1/p62 (p62/SQSTM1) could play an important role in mediating the link between the two pathways. Because of its ability to interact with polyubiquitin chains, p62/SQSTM1 was suggested to be a receptor that binds and delivers polyubiquitinated proteins to the two proteolytic pathways (7Shin J. Arch. Pharm. Res. 1998; 21: 629-633Crossref PubMed Scopus (117) Google Scholar). p62/SQSTM1 is a protein prone to aggregation and was first isolated in human tissues by Shin and co-workers (8Park I. Chung J. Walsh C.T. Yun Y. Strominger J.L. Shin J. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 12338-12342Crossref PubMed Scopus (90) Google Scholar). At its N terminus, p62/SQSTM1 has a Phox-BEM1 domain, which is a protein-protein interaction domain that assumes ubiquitin-like folding and can directly bind to proteasomes and other Phox-BEM1-containing proteins, including itself (9Wooten M.W. Hu X. Babu J.R. Seibenhener M.L. Geetha T. Paine M.G. Wooten M.C. J. Biomed. Biotechnol. 2006; 2006: 62079Crossref PubMed Scopus (96) Google Scholar). Recently, p62/SQSTM1 was shown to also interact with LC3, a protein that is an autophagosomal marker (10Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3380) Google Scholar). p62/SQSTM1 binds directly to LC3 via a 22-amino acid sequence, the LC3-interacting region (10Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3380) Google Scholar, 11Seibenhener M.L. Geetha T. Wooten M.W. FEBS. Lett. 2007; 581: 175-179Crossref PubMed Scopus (133) Google Scholar). By binding to polyubiquitinated proteins via its C-terminal ubiquitin-associated domain, to proteasomes via its N-terminal Phox-BEM1 domain, and to LC3-II via its LC3-interacting region domain, p62/SQSTM1 could direct polyubiquitinated proteins to the proteasome or to autophagy, when proteasomes are impaired or overwhelmed (10Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3380) Google Scholar, 11Seibenhener M.L. Geetha T. Wooten M.W. FEBS. Lett. 2007; 581: 175-179Crossref PubMed Scopus (133) Google Scholar). p62/SQSTM1 may thus be a candidate for the missing link between the UPP and autophagy. Here, we compared the dynamics of the turnover of ubiquitinated proteins by proteasomes and autophagy by assessing the effect of pharmacologic inhibitors of each pathway on the accumulation of endogenous ubiquitinated proteins as well as on p62/SQSTM1. We conclusively demonstrate that autophagy impairment does not cause the accumulation of ubiquitinated proteins. The minimal accumulation of ubiquitinated proteins observed upon chloroquine treatment is due to weak proteasome inhibition by the lysosomotropic agent. Other autophagy inhibitors or genetic impairment of autophagy in Atg5−/− MEFs did not cause the accumulation of ubiquitinated proteins. Furthermore, it is clear that p62/SQSTM1 is associated with proteasomes and is degraded mainly by autophagy. The role of p62/SQSTM1 in both proteolytic pathways, i.e. proteasomes and autophagy, requires further elucidation. The protease inhibitors used are as follows: chloroquine, bafilomycin A1, 3-methyladenine, and ammonium chloride (Sigma); epoxomicin (Peptides International Inc., Louisville, KY). The substrates used are as follows: Suc-LLVY-AMC (Bachem Bioscience Inc., King of Prussia, PA). The primary antibodies used are as follows: rabbit polyclonal anti-ubiquitinated proteins (1:1500, catalog no. Z0458, Dako North America, Carpinteria, CA); mouse monoclonal anti-ubiquitin antibody (for immunofluorescence detects mono- and polyubiquitinated proteins, 1:500, catalog no. BML-PW8810), anti-α4 (1:500, catalog no. PW8120), anti-Rpt6/S8 (1:1000, catalog no. PW9265), rabbit polyclonal anti-β5 (1:1000, catalog no. PW8895), in vitro synthesized Lys63-only and Lys48-only polyubiquitinated substrates (all from Enzo Life Sciences, Farmingdale, NY); mouse monoclonal anti-B-actin (1:10,000, catalog no. A-2228) and rabbit polyclonal anti-B-actin (1:10,000, catalog no. A-2066) both from Sigma; rabbit polyclonal anti-p62/SQSTM1 (1:1000, catalog no. PM045) and anti-Atg5 (1:1000, catalog no. PM050), and mouse monoclonal anti-Atg16 (1:1000, catalog no. M150–3) from MBL International Corp. (Woburn, MA). For the glycerol gradient fractionation the following were used: mouse monoclonal p62/SQSTM1 (Lck ligand) (1:500, catalog no. 610833, BD Transduction Laboratories, San Jose, CA); rabbit polyclonal anti-LC3 (1:1000, catalog no. Nb 100-2220, Novus Biological, Littleton, CO); secondary antibodies with HRP conjugate (1:10,000, Bio-Rad); and for immunofluorescence, Alexa-488 goat anti-mouse and Alexa-568 goat anti-rabbit (1:500, catalog no. A11029 and A11036, respectively, Molecular Probes, Carlsbad, CA). Human neuroblastoma SK-N-SH cells were derived from peripheral tissue (12Biedler J.L. Roffler-Tarlov S. Schachner M. Freedman L.S. Cancer Res. 1978; 38: 3751-3757PubMed Google Scholar) and were obtained from ATCC. Under serum conditions, the cells were maintained at 37 °C and 5% CO2 in minimal essential media with Eagle's salts containing 2 mm l-glutamine, 1 mm sodium pyruvate, 0.4% minimal essential media vitamins, 0.4% minimal essential media nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% normal fetal bovine serum. Under serum-free conditions, the cells were maintained as in serum conditions except that the media lacked nonessential amino acids and fetal bovine serum. Wild type and Atg5−/− mouse embryonic fibroblast cell lines (MEFs) were obtained from RIKEN BRC (Japan) and cultured at 37 °C and 5% CO2 in DMEM with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% normal fetal bovine serum as described previously (13Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2423) Google Scholar). The Atg5−/− cell line is deficient in Atg5, which is essential for autophagosome formation. Wild type and Atg5−/− MEFs were prepared from 13.5-day-old embryos and transformed with pEF321-T, an SV40 large T antigen expression vector to generate immortalized cell lines (13Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2423) Google Scholar). Rat cerebral cortical neuronal cultures were prepared from E18 embryos obtained from pregnant Sprague-Dawley females following the methods described previously (14Biederer T. Scheiffele P. Nat. Protoc. 2007; 2: 670-676Crossref PubMed Scopus (117) Google Scholar). Cells were cultured at 37 °C and 5% CO2 in neurobasal media supplemented with B27 and 0.5 mm l-GlutaMAX. Cells were plated on 100-mm dishes precoated with 50 μg/ml poly-d-lysine and at a density of 6 million cells per dish. Experiments were carried out following 8 days in culture. Cells were treated at 37 °C for different times with vehicle (0.5% DMSO) or different concentrations of the protease inhibitors listed above. Drugs were added dropwise directly into the medium with a gentle swirl of the culture plate. At the end of the incubation, all cultures were washed twice with phosphate-buffered saline (PBS) and processed for the different assays as described below. Cell washes removed unattached cells; therefore, subsequent assays were performed on adherent cells only. Cell survival was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously (15Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46884) Google Scholar). After treatment, cells were rinsed twice with PBS and harvested by gently scraping into hot (100 °C) SDS buffer (0.01 m Tris-EDTA, pH 7.5, and 1% SDS) to make sure all intracellular proteins were included. Samples were subjected to a 5-min boil at 100 °C followed by a brief sonication. After determination of the protein concentration with the bicinchoninic acid assay kit (Pierce), the following was added to each sample: β-mercaptoethanol (358 mm), bromphenol blue (0.005%), glycerol (20%), and SDS (4%) in stacking gel buffer (0.1 m Tris-Cl, pH 6.8). Following SDS-PAGE on 8 or 10% polyacrylamide gels, proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were probed with the respective antibodies, and antigens were visualized by a standard chemiluminescent horseradish peroxidase method with the ECL reagent. Semi-quantification of protein detection was done by image analysis with the ImageJ program (National Institutes of Health, //rsb.info.nih.gov). Relative intensity (no units) is the ratio between the value for each protein and the value for the respective loading control. Total cell lysates were prepared on ice by homogenization in 0.01 m Tris-EDTA, pH 7.5, buffer. The lysates were cleared by a 15-min centrifugation (19,000 × g) at 4 °C. The cleared samples were normalized for protein concentration determined with the bicinchoninic acid assay kit (Pierce). The chymotrypsin-like activity was assayed colorimetrically in 25 μg of protein/sample with the substrate Suc-LLVY-AMC (400 μm in DMSO) after 24-h incubations at 37 °C as described previously (16Wilk S. Orlowski M. J. Neurochem. 1983; 40: 842-849Crossref PubMed Scopus (288) Google Scholar). Values obtained were in the linear range of the reaction (data not shown). Cells were harvested in 25 mm Tris-HCl, pH 7.5, 2 mm ATP, and 1 mm DTT. Following homogenization and sonication, the lysates were centrifuged (19,000 × g for 15 min) at 4 °C. The cleared supernatants (2 mg of protein/sample) were subjected to centrifugation (83,000 × g for 24 h) at 4 °C in a Beckman SW41 rotor in a 10–40% glycerol gradient (fractions 13 to 1) made in the same lysis buffer. Following centrifugation, 13 fractions (800 μl each) were collected and analyzed. Aliquots (50 μl) of each fraction were assayed for chymotrypsin-like activity with the substrate Suc-LLVY-AMC colorimetrically after 24-h incubations at 37 °C as described previously (16Wilk S. Orlowski M. J. Neurochem. 1983; 40: 842-849Crossref PubMed Scopus (288) Google Scholar). Proteins were precipitated with acetone from 700 μl of each fraction and subjected to Western blot analysis (10% gels). The membranes were probed with the respective antibodies, and antigens were visualized by a standard chemiluminescent horseradish peroxidase method with the ECL reagent. Upon treatment with vehicle (DMSO) or the respective drugs, cells were washed twice with PBS and were harvested with the following buffer A: 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 5 mm ATP (grade 1; Sigma), 1 mm DTT and 10% glycerol, which preserves 26 S proteasome assembly (17Elsasser S. Schmidt M. Finley D. Methods Enzymol. 2005; 398: 353-363Crossref PubMed Scopus (135) Google Scholar). Following homogenization and centrifugation (19,000 × g for 15-min) at 4 °C, the protein concentration of the cleared supernatants was determined with the Bradford assay (Bio-Rad) and normalized with buffer A. The cleared supernatants (80 μg of protein/lane for proteasome activity and 40 μg of protein/lane for Western blotting) were resolved by nondenaturing PAGE using a modification of the method described previously (18Ogburn K.D. Figueiredo-Pereira M.E. J. Biol. Chem. 2006; 281: 23274-23284Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We used a gel consisting of three layers of equal amounts, from the bottom up, of 5, 4, and 3% polyacrylamide with RhinohideTM polyacrylamide strengthener (Molecular Probes). Bromphenol blue was added to the protein samples prior to loading. Nondenaturing minigels were run at 150 V for either 3 h or 90 min. The gels were then incubated on a rocker for 10 min at 37 °C with 15 ml of 400 μm Suc-LLVY-AMC in buffer B (buffer A modified to contain 1 mm ATP). Proteasome bands were visualized upon exposure to UV light (360 nm) and were photographed with a NIKON Cool Pix 8700 camera with a 3-4219 fluorescent green filter (Peca Products, Inc.). Proteins on the native gels were transferred (110 mA) for 2 h onto PVDF membranes. Western blot analyses were then carried out sequentially for detection of the 20 S and 26 S proteasomes with anti-β5 and anti-Rpt6/S8 subunit antibodies. The anti-β5 antibody reacts with a core particle subunit and therefore detects both the 26 S and 20 S proteasomes. The anti-Rpt6/S8 antibody reacts with a regulatory particle subunit, thus only detecting 26 S proteasomes. Antigens were visualized by a chemiluminescent horseradish peroxidase method with the ECL reagent. Aliquots of the samples were also boiled for 5 min in Laemmli buffer and loaded onto 10% gels (40 μg of protein/lane) for Western blot analysis following SDS-PAGE. Cells were washed twice with PBS and harvested with RIPA buffer (20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 1 mm EGTA, 10% glycerol (v/v), 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm β-glycerophosphate, 2.5 mm sodium pyrophosphate, 50 mm sodium fluoride, 1% Nonidet P-40, and protease inhibitor mixture). Following homogenization, lysates were centrifuged at 4 °C (for 15 min at 19,000 × g). Cell pellets were resuspended by sonication in harvesting buffer containing 2% SDS; 100 μg of protein/sample were trapped by filtration through a pre-wet Trans-Blot nitrocellulose membrane 0.2 μm adapted to a 96-well dot blot apparatus (Bio-Rad) as described previously (19Wanker E.E. Scherzinger E. Heiser V. Sittler A. Eickhoff H. Lehrach H. Methods Enzymol. 1999; 309: 375-386Crossref PubMed Scopus (201) Google Scholar). Because of the 0.2-μm pore size of this membrane, only aggregated proteins are retained, although the soluble ones pass through the pores of the membrane. To detect aggregates, the membranes were probed with the anti-p62/SQSTM1 or the anti-ubiquitinated protein antibodies. After treatment, SK-N-SH cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The PLA was performed as described previously (20Söderberg O. Gullberg M. Jarvius M. Ridderstråle K. Leuchowius K.J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.G. Landegren U. Nat. Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1786) Google Scholar) and with the Duolink PLA detection kit 613 from Axxora, LLC, San Diego. Slides were mounted with Vectashield medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA). Cell staining was visualized with an UltraViewVoX spinning disk confocal microscope (PerkinElmer Life Sciences). After treatment, SK-N-SH cells were fixed for 30 min in 4% paraformaldehyde and post-fixed for 2 min in ice-cold methanol, followed by blocking and permeabilization in 2.5% BSA, 5% normal goat serum, and 0.3% Triton X-100 for 1 h, and co-incubated overnight at 4 °C with the respective antibodies. Slides were mounted with ProLong® Gold antifade mounting reagent with DAPI (Invitrogen). Cell staining was visualized with an Axio Imager M2 microscope (Carl Zeiss Micro Imaging, Inc. Thornwood, NY). statistical significance was estimated using one-way analysis of variance (Tukey-Kramer multiple comparison test) with the Instat 2.0, Graphpad software (San Diego). To investigate the link between autophagy and degradation of ubiquitinated proteins, SK-N-SH cells were treated with three different autophagy inhibitors, namely chloroquine (CQ, 100 μm), ammonium chloride (NH4Cl, 10 mm), and bafilomycin A1 (200 nm) at concentrations reported in the literature to inhibit autophagy. We compared the effects of the three drugs on cell viability and autophagy inhibition. In parallel studies, we treated cells with epoxomicin (Epx, 25 nm), an irreversible proteasome inhibitor. Fig. 1A shows that at the concentrations tested bafilomycin A1 was the most toxic of the drugs tested. Autophagy inhibition was assessed by accumulation of the autophagosome marker LC3-II, which is accepted as an overall indicator of autophagy impairment. LC3-II levels were most prominent after chloroquine treatment (123-fold increase) as indicated by the strong LC3-II band on the Western blots (Fig. 1B). In bafilomycin A1-treated cells, LC3-II levels (32-fold increase) were lower than in chloroquine-treated cells, and ammonium chloride and epoxomicin treatment failed to induce LC3-II accumulation (Fig. 1B). These data show that among the three autophagy inhibitors tested, chloroquine most effectively raised LC3-II levels. Proteasome inhibition by epoxomicin had no inhibitory effect on autophagy. p62/SQSTM1 was suggested to be specifically degraded by autophagy (10Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3380) Google Scholar, 21Komatsu M. Waguri S. Koike M. Sou Y.S. Ueno T. Hara T. Mizushima N. Iwata J. Ezaki J. Murata S. Hamazaki J. Nishito Y. Iemura S. Natsume T. Yanagawa T. Uwayama J. Warabi E. Yoshida H. Ishii T. Kobayashi A. Yamamoto M. Yue Z. Uchiyama Y. Kominami E. Tanaka K. Cell. 2007; 131: 1149-1163Abstract Full Text Full Text PDF PubMed Scopus (1742) Google Scholar), and its levels were proposed to increase in response to autophagy inhibition (22Bjørkøy G. Lamark T. Brech A. Outzen H. Perander M. Overvatn A. Stenmark H. Johansen T. J. Cell Biol. 2005; 171: 603-614Crossref PubMed Scopus (2547) Google Scholar). We compared how the four proteolytic inhibitors listed above affected p62/SQSTM1 as well as ubiquitinated protein levels. It is clear that from the four drugs tested, epoxomicin most effectively increased p62/SQSTM1 (9-fold, Fig. 1C) and ubiquitinated protein (3-fold, Fig. 1D) levels in SK-N-SH cells. Chloroquine caused a detectable increase in p62/SQSTM1 (6-fold), albeit to a lesser extent than epoxomicin. Ammonium chloride and bafilomycin A did not increase p62/SQSTM1 levels. Another autophagy inhibitor, 3-MA, failed to increase the levels of ubiquitinated proteins, p62/SQSTM1 and LC3-II (supplemental Fig. 1). Upon treatment with chloroquine, we observed a 1.7-fold increase in ubiquitinated proteins. Actin levels were not increased by any of the treatments (Fig. 1E). To further investigate the cross-talk between the UPP and autophagy, we decided to focus our studies on chloroquine and epoxomicin, because ammonium chloride and bafilomycin A1 failed to increase p62/SQSTM1 and ubiquitinated protein levels, and the latter inhibitor was highly cytotoxic. We compared the effects of inhibiting each pathway alone with inhibiting both pathways together (Fig. 2). Similar results were obtained in cells maintained under serum or serum-free conditions (the latter not shown). p62/SQSTM1 levels were raised 4-fold over control by epoxomicin (p < 0.01) but only 2-fold by chloroquine (p > 0.05, not significant) (Fig. 2, A and D). In cells treated with both inhibitors, p62/SQSTM1 levels were higher than in cells treated with epoxomicin alone, but the difference was not statistically significant (p > 0.05). As expected, LC3-II levels were increased only in cells treated with chloroquine as proteasome inhibition does not block autophagy function. Chloroquine alone raised LC3-II levels 2-fold (p < 0.01, Fig. 2, B and D). LC3-II levels were similar in cells treated with chloroquine alone or in combination with epoxomicin (p > 0.05). The levels of LC3-I and the α4 subunit of the 20 S proteasome did not show any significant changes (Fig. 2, B–D). Together, these data clearly demonstrate that proteasome inhibition most efficiently increases p62/SQSTM1 levels when compared with autophagy impairment, the latter ascertained by accumulation of LC3-II in cells treated with chloroquine. Inhibition of lysosomal function was recently shown to reduce proteasome activity (23Qiao L. Zhang J. Neurosci. Lett. 2009; 456: 15-19Crossref PubMed Scopus (43) Google Scholar), and therefore, we compared the effect of epoxomicin and chloroquine on the cleavage of Suc-LLVY-AMC, a short substrate used to measure proteasome activity. Similar results were obtained under serum and serum-free conditions (the latter not shown). As shown in Fig. 2G, Suc-LLVY-AMC hydrolysis measured in total lysates was blocked by at least 80% in cells treated with epoxomicin or chloroquine alone or combined. Despite a similar decline in Suc-LLVY-AMC hydrolysis observed in cells treated with each or both drugs, the levels of ubiquitinated proteins were quite different. Treatment with epoxomicin caused an ∼3-fold rise in ubiquitinated protein levels (p < 0.01) compared with a much smaller increase (1.8-fold, p > 0.05) in cells treated with chloroquine (Fig. 2, E and F). Combined treatment with epoxomicin and chloroquine enhanced ubiquitinated protein levels by 4-fold, which was significantly different from the values observed under epoxomicin alone (p < 0.01, Fig. 2, E and F). Ubiquitin protein aggregates were detected with the filter trap assay in cells treated with epoxomicin by itself or in combination with chloroquine (Fig. 2H). Actin levels were not changed upon proteasome and/or autophagy inhibition (Fig. 2E). These data clearly demonstrate that measuring Suc-LLVY-AMC hydrolysis using total cell lysates does not accurately reflect proteasome activity. This is not surprising, because it is well established that the substrate Suc-LLVY-AMC is cleaved not only by the proteasome (24Stein R.L. Melandri F. Dick L. Biochemistry. 1996; 35: 3899-3908Crossref PubMed Scopus (116) Google Scholar) but also by other chymotrypsin-like proteases as well as by calpains (25Sasaki T. Kikuchi T. Yumoto N. Yoshimura N. Murachi T. J. Biol. Chem. 1984; 259: 12489-12494Abstract Full Text PDF PubMed Google Scholar). In addition, it is clear that the overall levels of ubiquitinated proteins in chloroquine-treated cells are very low. This includes Lys48- and Lys63-linked chains as both are detected with the polyubiquitin antibody from DAKO (Carpinteria, CA) used in these studies (Fig. 2E, left panel). This is relevant to autophagy because Lys63-linked chains were postulated to selectively facilitate the clearance of ubiquitinated proteins via the autophagic pathway (26Tan J.M. Wong E.S. Kirkpatrick D.S. Pletnikova O. Ko H.S. Tay S.P. Ho M.W. Troncoso J. Gygi S.P. Lee M.K. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2008; 17: 431-439Crossref PubMed Scopus (330) Google Scholar). The decline in Suc-LLVY-AMC hydrolysis together with the accumulati" @default.
• W2018082393 created "2016-06-24" @default.
• W2018082393 creator A5002567720 @default.
• W2018082393 creator A5012719746 @default.
• W2018082393 date "2011-06-01" @default.
• W2018082393 modified "2023-09-29" @default.
• W2018082393 title "Dynamics of the Degradation of Ubiquitinated Proteins by Proteasomes and Autophagy" @default.
• W2018082393 cites W1490867178 @default.
• W2018082393 cites W1492667719 @default.
• W2018082393 cites W1523373796 @default.
• W2018082393 cites W1560487523 @default.
• W2018082393 cites W1602776657 @default.
• W2018082393 cites W1964976537 @default.
• W2018082393 cites W1976066524 @default.
• W2018082393 cites W1978646107 @default.
• W2018082393 cites W1981502202 @default.
• W2018082393 cites W1981783595 @default.
• W2018082393 cites W1986083613 @default.
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• W2018082393 cites W1991558827 @default.
• W2018082393 cites W1991915276 @default.
• W2018082393 cites W1992496728 @default.
• W2018082393 cites W1992659717 @default.
• W2018082393 cites W1993801059 @default.
• W2018082393 cites W1994602517 @default.
• W2018082393 cites W1997112179 @default.
• W2018082393 cites W1999795482 @default.
• W2018082393 cites W2005997790 @default.
• W2018082393 cites W2006398507 @default.
• W2018082393 cites W2007442181 @default.
• W2018082393 cites W2012040176 @default.
• W2018082393 cites W2014918593 @default.
• W2018082393 cites W2022812113 @default.
• W2018082393 cites W2031612618 @default.
• W2018082393 cites W2032124050 @default.
• W2018082393 cites W2037417750 @default.
• W2018082393 cites W2039137539 @default.
• W2018082393 cites W2040337522 @default.
• W2018082393 cites W2046879630 @default.
• W2018082393 cites W2077731318 @default.
• W2018082393 cites W2082788676 @default.
• W2018082393 cites W2087920955 @default.
• W2018082393 cites W2092956762 @default.
• W2018082393 cites W2095894333 @default.
• W2018082393 cites W2114918609 @default.
• W2018082393 cites W2119399459 @default.
• W2018082393 cites W2124975942 @default.
• W2018082393 cites W2128537698 @default.
• W2018082393 cites W2135574023 @default.
• W2018082393 cites W2146124002 @default.
• W2018082393 cites W2148085004 @default.
• W2018082393 cites W2149761960 @default.
• W2018082393 cites W2154602568 @default.
• W2018082393 cites W2155738133 @default.
• W2018082393 cites W2157940538 @default.
• W2018082393 cites W2158892689 @default.
• W2018082393 cites W2165453045 @default.
• W2018082393 cites W2166415858 @default.
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