HspB8 Chaperone Activity toward Poly(Q)-containing Proteins Depends on Its Association with Bag3, a Stimulator of Macroautophagy
2007; Elsevier BV; Volume: 283; Issue: 3 Linguagem: Inglês
10.1074/jbc.m706304200
ISSN1083-351X
AutoresSerena Carra, Samuel J. Seguin, Herman Lambert, Jacques Landry,
Tópico(s)Heat shock proteins research
ResumoMutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy. Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy. The intracellular aggregation of proteins, whether it occurs as a result of proteotoxic stress or genetic mutations, represents a major threat in the crowded environment of the cell. Consequently, efficient mechanisms of protein quality control exist involving molecular chaperones such as Hsp70 and Hsp90, which can recognize and bind to unfolded proteins thereby preventing their aggregation (1Lindquist S. Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar, 2Esser C. Alberti S. Hohfeld J. Biochim. Biophys. Acta. 2004; 1695: 171-188Crossref PubMed Scopus (198) Google Scholar, 3Barral J.M. Broadley S.A. Schaffar G. Hartl F.U. Semin. Cell Dev. Biol. 2004; 15: 17-29Crossref PubMed Scopus (250) Google Scholar, 4Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (941) Google Scholar). The fate of the chaperone substrates is determined by associated co-chaperones. CHIP, a ubiquitin E3 ligase and Bag1, a member of the Bag family of proteins (Bag1-Bag6) that contains a ubiquitin-like domain, associate with Hsp70 and facilitate the targeting of Hsp70 substrates to the ubiquitin-proteasome pathway (5Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Hohfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 6Demand J. Alberti S. Patterson C. Hohfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). In contrast, by associating with Hsp70, Bag2 hampers CHIP activity and favors substrate renaturation (7Dai Q. Qian S.B. Li H.H. McDonough H. Borchers C. Huang D. Takayama S. Younger J.M. Ren H.Y. Cyr D.M. Patterson C. J. Biol. Chem. 2005; 280: 38673-38681Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 8Arndt V. Daniel C. Nastainczyk W. Alberti S. Hohfeld J. Mol. Biol. Cell. 2005; 16: 5891-5900Crossref PubMed Scopus (157) Google Scholar), whereas Bag3 prevents Hsp70 substrate degradation and causes ubiquitylated substrate accumulation (9Doong H. Rizzo K. Fang S. Kulpa V. Weissman A.M. Kohn E.C. J. Biol. Chem. 2003; 278: 28490-28500Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The role of chaperone complexes containing Hsp70 has been experimentally highlighted in a range of neurodegenerative disorders characterized by the accumulation of protein aggregates (10Wyttenbach A. Carmichael J. Swartz J. Furlong R.A. Narain Y. Rankin J. Rubinsztein D.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2898-2903Crossref PubMed Scopus (306) Google Scholar, 11Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M. Bonini N.M. Science. 2002; 295: 865-868Crossref PubMed Scopus (1065) Google Scholar, 12Cummings C.J. Sun Y. Opal P. Antalffy B. Mestril R. Orr H.T. Dillmann W.H. Zoghbi H.Y. Hum. Mol. Genet. 2001; 10: 1511-1518Crossref PubMed Scopus (423) Google Scholar, 13Muchowski P.J. Wacker J.L. Nat. Rev. Neurosci. 2005; 6: 11-22Crossref PubMed Scopus (816) Google Scholar, 14Bonini N.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16407-16411Crossref PubMed Scopus (180) Google Scholar). However, it is within the HspB group of heat shock proteins (also known as small heat shock proteins) that mutations have first been associated with human diseases (15Benndorf R. Welsh M.J. Nat. Genet. 2004; 36: 547-548Crossref PubMed Scopus (56) Google Scholar). We previously showed that HspB8 (H11/Hsp22), a member of this family, has chaperone activity in vivo toward Htt43Q, a pathogenic form of huntingtin that contains an expanded polyglutamine stretch making the protein prone to aggregation. Overexpression of HspB8 but not two closely related family members, HspB1 (Hsp27) or HspB5 (αB-crystallin), accelerated the degradation of Htt43Q and prevented both aggregation and formation of inclusion bodies (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar). A similar chaperone activity was observed using as substrate the androgen receptor protein containing a 65 glutamine extension (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar), suggesting that this chaperone activity could at least be generalized to polyglutamine-containing proteins. Here, we have investigated the mechanism of action of HspB8. We found that HspB8 forms a stable stoichiometric complex with the co-chaperone Bag3. We further show that Bag3 stimulates macroautophagy, thus facilitating the degradation of mutated huntingtin. Plasmids and Reagents—pHDQ43-HA 3The abbreviations used are: HAhemagglutinin3-MA3-methyladenineHspheat shock proteinsiRNAsmall interfering RNA. encoding a HA-tagged Huntingtin exon 1 fragment with 43 CAG repeats was from Dr. Rubinsztein (10Wyttenbach A. Carmichael J. Swartz J. Furlong R.A. Narain Y. Rankin J. Rubinsztein D.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2898-2903Crossref PubMed Scopus (306) Google Scholar). Plasmids encoding human HspB8 and myc-tagged HspB8, Hsp70, and HspB1, were described previously (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar, 17Chávez Zobel A.T. Loranger A. Marceau N. Thériault J.R. Lambert H. Landry J. Hum. Mol. Genet. 2003; 12: 1609-1620Crossref PubMed Scopus (135) Google Scholar). The plasmid encoding His-tagged human Bag3 (pCINHisBag3) was from Dr. Kohn (9Doong H. Rizzo K. Fang S. Kulpa V. Weissman A.M. Kohn E.C. J. Biol. Chem. 2003; 278: 28490-28500Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). pGFP-LC3 and pMyc-LC3 were a kind gift of Dr. T. Yoshimori (18Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar). 3-Methyladenine (3-MA), wortmannin, Pepstatin, and E64d were from Sigma. hemagglutinin 3-methyladenine heat shock protein small interfering RNA. Antibodies—Anti-HspB8 and anti-Hu27 are rabbit polyclonal antibodies against human HspB8 and HspB1, respectively (17Chávez Zobel A.T. Loranger A. Marceau N. Thériault J.R. Lambert H. Landry J. Hum. Mol. Genet. 2003; 12: 1609-1620Crossref PubMed Scopus (135) Google Scholar). Anti-LC3 was developed in rabbit against a recombinant protein made of glutathione S-transferase fused to rat LC3 lacking the 22 C-terminal residues (ending at Gly120, the hypothetical cleavage site in LC3-II) (18Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar). Anti-Hsc70 and anti-Hsp70 were from Santa Cruz and Stressgen Bioreagents, respectively. Anti-HA (HA.11) and anti-γ-tubulin were from Sigma, anti-myc (9E10) was from American Type Culture Collection, and anti-Bag1 was from BIOSOURCE. Anti-Bag3 was from GeneTex except for the data of Fig. 1b, where an in-house antibody was used, which was raised against the recombinant glutathione S-transferase-human Bag3 fusion protein. Cell Culture, Transfection, and Immunofluorescence Cell Staining—All cell lines were grown in Dulbecco's modified Eagle's medium with high glucose (Invitrogen). HeLa (human cervical cancer), HEK-293T (human embryonal kidney), and COS-1 (African green monkey kidney) cells were grown in medium supplemented with 10% fetal bovine serum, whereas 5% fetal bovine serum was used for CCL39 (Chinese hamster lung fibroblast) cells and 10% bovine calf serum was used for NIH3T3 (mouse embryonic fibroblast) cells. Cells were transfected by calcium phosphate precipitation as previously described (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar). Transfection of siRNA for HspB8 (target sequence: agagcaguuucaacaacga), HspB1 (target sequence: ugagacugccggcaaguaa), Bag3 (target sequence: gcaugccagaaaccacuca), and a control sequence (Dharmacon's siCONTROL non-targeting siRNA) were performed using Lipofectamine (Invitrogen), according to the manufacturer's instructions. Immunocytochemistry was performed as previously described (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar), except that the cells were fixed with 100% methanol for 30 min at -20 °C. For statistical analysis samples from three independent experiments were analyzed for each condition using the one-way analysis of variance test. l-[35S]Methionine Metabolic Labeling and Co-immunoprecipitation—HeLa cells were incubated overnight in minimum essential medium (Invitrogen) containing 4% fetal bovine serum and 15 μCi/ml of l-[35S]methionine (Amersham Biosciences). Cells were lysed in a buffer containing: 20 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2, 100 mm KCl, 0.5% Nonidet P-40, 3% glycerol, 1 mm dithiothreitol, complete EDTA-free (Roche). The cell lysates were centrifuged and cleared with protein A-Sepharose beads at 4 °C for 1 h. Protein A-Sepharose beads complexed with anti-HspB8 antibody or non-immune rabbit serum were added to the pre-cleared lysates. After incubation for 2 h at 4 °C, the immune complexes were centrifuged and the supernatants (output) collected. Beads were washed 4 times with the lysis buffer; both co-immunoprecipitated proteins and output fractions were resolved on SDS-PAGE. Dried polyacrylamide gel was exposed overnight using a Molecular Dynamic Storage Phosphor Screen (Kodak). Co-immunoprecipitations from non-labeled HeLa cells were performed as described above. Tandem Mass Spectrometry—Immunoprecipitates from HeLa cells using anti-HspB8 antibody were separated by SDS-PAGE and stained with Coomassie Blue R-250. The polypeptides migrating at ∼70-80 kDa were excised from the gel and send to the Southern Alberta Mass Spectrometry Centre for Proteomics (University of Calgary) for analyses by liquid chromatography-tandem mass spectrometry, using an ESI-TRAP instrument. Filter Trap Assay and SDS-soluble and -insoluble Cell Extracts—SDS-insoluble proteins were analyzed either by filter trap assay or PAGE. For filter trap assay, the cells were scraped and homogenized in 2% SDS filter trap assay buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 50 mm dithiothreitol). Four different dilutions of each sample were heated at 98 °C for 3 min and applied with mild suction onto a cellulose acetate membrane. The membrane was washed with 0.1% SDS filter trap assay buffer and processed for Western blot analysis. For PAGE analysis of SDS-soluble and SDS-insoluble proteins, the cells were scraped, homogenized, and heated for 10 min at 100 °C in 2% SDS sample buffer supplemented with 50 mm dithiothreitol. The samples were centrifuged for 20 min at room temperature and the supernatants were used as the SDS-soluble fraction. The pellets (SDS-insoluble fraction) were incubated with 100% formic acid for 30 min at 37 °C, lyophilized overnight, and finally resuspended in the same volume of 2% SDS sample buffer as the SDS-soluble fraction. Both SDS-soluble and SDS-insoluble fractions were processed for Western blotting. Glycerol Gradient Centrifugation—NIH 3T3 cell extracts were prepared 24 h after transfection by sonication in 1 ml of a 3.33% glycerol HEPES buffer (25 mm HEPES, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, pH 7.4). After centrifugation at 18,000 × g for 10 min at 4 °C, the supernatants were loaded on top of a 11-ml linear gradient of glycerol (10-40%) made in HEPES buffer. The tubes were centrifuged for 16 h at 34,000 × g in a SW40 rotor (Beckman) at 4 °C. The gradients were fractionated and the distribution of HspB8 and Bag3 was analyzed by Western blotting. The gradient was calibrated from the positions of known protein complexes (19Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). HspB8 Forms a Stable Stoichiometric Complex with Bag3—Upon expression in cells, poly(Q) proteins such as Htt43Q forms small aggregates all over the cytoplasm, which gradually become SDS-insoluble and fuse to each other to form large amorphous inclusion bodies that accumulate in the juxtanuclear area of the cells (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar). We previously described that the overexpression of HspB8 prevented the accumulation of HA-tagged Htt43Q (HA-Htt43Q), presumably by accelerating its degradation. This effect was not observed when overexpressing two other members of the HspB family, HspB1 or HspB5. In an initial attempt to determine the mechanism of action of HspB8 as a molecular chaperone, we investigated whether functional partners of HspB8 could be identified. Extracts prepared from HeLa cells metabolically labeled with [35S]methionine were immunoprecipitated with specific anti-HspB8 antibodies. A protein with an apparent relative molecular mass of 80,000 abundantly co-immunoprecipitated with HspB8 and was identified as Bag3 by mass spectrometry (Fig. 1a). Co-immunoprecipitation experiments followed by Western blots performed in HeLa cells (Fig. 1b) and also in COS-1 cells (data not shown) confirmed the specificity of the interaction between HspB8 and Bag3. Bag3, but not Bag1, was co-immunoprecipitated by HspB8 antibodies, whereas HspB8, but not HspB1, was co-immunoprecipitated with Bag3 antibodies. HspB8 and Bag3 contain 5 and 10 methionine residues, respectively. From the amount of radioactivity associated with each of the two immunoprecipitated proteins, a stoichiometric ratio of 2:1 was determined for the HspB8-Bag3 complex. The complex was further characterized by glycerol gradient analysis in NIH3T3 cells, a cell line used here because it expresses very little endogenous HspB8. When expressed at high concentrations (transfecting 3 μg of plasmid), HspB8 sedimented with an apparent molecular mass of about 50 kDa, as expected for a homodimer, the basic building unit of the HspB protein family (Fig. 1c) (19Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). However, when expressed at lower concentrations (transfecting 0.3 μg of plasmid), a portion of the protein multimerized into large complexes sedimenting between the 62- and 350-kDa markers, consistent with the molecular mass of 130 kDa predicted for the 2:1 HspB8-Bag3 complex. This suggested that upon transfection of HspB8, endogenous Bag3 becomes limiting for the formation of the HspB8-Bag3 complex. In agreement with this idea, co-expressing Bag3 with HspB8 allowed a higher proportion of HspB8 to incorporate into the approximate 130-kDa complex (Fig. 1c). We also determined that the presence of Bag3 was important for the stability of HspB8. In CCL39 and HeLa cells, overexpressing Bag3 together with HspB8 yielded a much higher expression of HspB8 than overexpressing HspB8 alone or HspB8 with Hsp70 (taken as control) (Fig. 1d). This was not caused by an increased transcriptional activity because HspB8 mRNA levels were not affected by Bag3 overexpression (supplemental Fig. S1). These findings suggested that the HspB8 proteins may be less stable when Bag3 is limiting. Accordingly, knocking down Bag3 expression by 75% using siRNA resulted in a 75% decrease in endogenous HspB8 expression, without affecting the expression of HspB1 (Fig. 1e). In contrast, reducing the HspB8 concentration below detectable levels caused little reduction in Bag3 (Fig. 1f). As an additional control, knocking down HspB1 caused no significant change in the expression level of either HspB8 or Bag3 (Fig. 1g). The reduction of HspB8 endogenous levels following treatment with Bag3 siRNA was observed not only in HeLa cells, where the complex has been first identified, but also in HEK-293T cells (see Fig. 4). All together these observations suggest that in different cell types, most HspB8 is associated with a pool of Bag3 and that this complex contributes importantly to HspB8 stability. Bag3 Also Prevents Htt43Q Accumulation—We next investigated whether overexpression of Bag3 in cultured cells, like HspB8, prevents the accumulation of the polyglutamine protein Htt43Q. Cells were transfected with vectors encoding HA-Htt43Q alone or HA-Htt43Q and either Myc-HspB8, His-tagged Bag3 or both. The accumulation of Htt43Q aggregates was analyzed 44 h after transfection using the filter trap assay (filtration of the SDS lysates through a cellulose acetate membrane, which entraps the aggregates). Expression of Bag3, like HspB8, but not expression of Hsp70, dramatically reduced the accumulation of insoluble Htt43Q in both CCL39 cells (Fig. 2a), the cell line where the activity of HspB8 was initially characterized (16Carra S. Sivilotti M. Chavez Zobel A.T. Lambert H. Landry J. Hum. Mol. Genet. 2005; 14: 1659-1669Crossref PubMed Scopus (149) Google Scholar) and in HeLa cells (Fig. 2b). Similar results were also obtained in HEK-293T and COS-1 cells (see Figs. 3, 4, 6, and supplemental Fig. S2), suggesting that the activity was cell line-independent. Notably, an additive effect was observed when HspB8 and Bag3 were expressed together (Fig. 2, a and b). In control experiments in which green fluorescent protein was co-transfected with mutated huntingtin, HspB8 or Bag3 produced no reduction in the percentage of cells expressing green fluorescent protein nor in the survival of these cells, indicating that the reduction of HA-Htt43Q expression was not a consequence of toxicity (data not shown).FIGURE 3HspB8 requires Bag3 to efficiently prevent the accumulation of Htt43Q. HEK-293T cells were either sham-treated (-) or treated (+) with a 50 nm solution of control siRNA (siRNA con.), siRNA targeting Bag3 (siRNA Bag3), or siRNA targeting HspB8 (siRNA HspB8). Twenty-four h later, the cells were transfected with plasmids encoding HA-Htt43Q together with an empty vector (-), Myc-HspB8, or His-tagged Bag3 (+). SDS-soluble and SDS-insoluble fractions were prepared 44 h post-transfection. HA-Htt43Q levels were analyzed in both the SDS-soluble and SDS-insoluble fractions. The empty arrowheads show HA-Htt43Q migrating at ∼21 kDa. The efficiency of the siRNA treatments or transfections were measured by probing in the SDS-soluble fraction the levels of endogenous Bag3 (endo-Bag3), endogenous HspB8 (endo-HspB8), transfected HspB8 (myc-HspB8), transfected Bag3 (his-Bag3), and γ-tubulin (a and b).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Basal and HspB8/Bag3-stimulated degradation of Htt43Q is regulated by macroautophagy. a, HEK-293T and COS1 cells were transfected with the plasmid encoding HA-Htt43Q. Twelve h post-transfection, cells were treated with 10 mm 3-MA and 0.2 μm wortmannin (+) for 30 h or left untreated (-). SDS-insoluble extracts were then prepared and analyzed by Western blotting using the anti-HA antibody. b, COS1 cells were transfected with vectors encoding HA-Htt43Q and an empty vector (-), MycHspB8 (+HspB8), or HisBag3 (+Bag3). Fourteen h post-transfection, the cells were left untreated (w/o 3-MA and Wort.) or treated with 10 mm 3-MA and 0.2 μm wortmannin (with 3-MA and Wort.) for 30 h. SDS-soluble and SDS-insoluble fractions were separated and analyzed by Western blotting using the anti-HA and the γ-tubulin antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HspB8 Requires Bag3 to Exert Its Full Chaperone Activity—We then determined whether Bag3 was required for the activity of HspB8. HEK-293T cells were transfected with a control siRNA sequence or with a specific siRNA directed against Bag3. Twenty-four hours later, the cells were transfected with vectors encoding HA-Htt43Q alone or with myc-HspB8. Cells were incubated for an additional 48 h before being analyzed for the presence of SDS-soluble and SDS-insoluble HA-Htt43Q. Knocking down Bag3 expression efficiently blocked the ability of HspB8 to prevent Htt43Q accumulation (Fig. 3a). This was not due to a decreased expression of myc-HspB8 after the Bag3 siRNA treatment, thus suggesting that HspB8 requires Bag3 to exert its full chaperone function. A similar experiment was performed to investigate the impact of HspB8 knock-down on Bag3 activity. Fig. 3b shows that overexpressed Bag3 can block Htt43Q accumulation also in the absence of HspB8. All together these results suggested that Bag3 is an essential element of the HspB8-Bag3 complex and is responsible for the activity ascribed to HspB8 in decreasing Htt43Q accumulation. Decreasing Endogenous Bag3 Expression Resulted in Enhanced Htt43Q Aggregation—Both Bag3 and HspB8 are up-regulated upon proteotoxic stress such as exposures to heavy metal or heat shock (20Pagliuca M.G. Lerose R. Cigliano S. Leone A. FEBS Lett. 2003; 541: 11-15Crossref PubMed Scopus (101) Google Scholar, 21Chowdary T.K. Raman B. Ramakrishna T. Rao C.M. Biochem. J. 2004; 381: 379-387Crossref PubMed Scopus (111) Google Scholar). Therefore, HspB8 and/or Bag3 overexpression experiments mimic well the cellular stress response activated by misfolded proteins and aimed at their clearance. From the data presented in Fig. 3, however, it seemed that knocking down the expression of Bag3 or HspB8 alone had no significant effect on the accumulation of HA-Htt43Q. To better evaluate the role of endogenous HspB8 and Bag3 on Htt43Q accumulation, we repeated the experiment transfecting HEK-293T cells with a lower amount of cDNA encoding for HA-Htt43Q. Fig. 4a shows that pre-treatment with Bag3 and HspB8 siRNA together caused a significant increase in the amount of SDS-insoluble HA-Htt43Q as compared with cells transfected with control siRNA. This effect was quantified in two independent experiments and corresponded to a 2-fold increase in the amount of SDS-insoluble Htt43Q. Treatments with Bag3 siRNA alone (which, as described in Fig. 1e, also induced the depletion of HspB8) resulted in a similar increase of SDS-insoluble HA-Htt43Q. In contrast, depleting HspB8 alone had no effect. These results strongly suggested that Bag3 plays an active role in the basal capacity of the cells to eliminate badly folded proteins as they are produced. Whether or not Bag3 does it as part of the HspB8-Bag3 complex and uses alternative partners when HspB8 levels are decreased remains to be determined. Htt43Q Accumulation in Cells Depleted for Bag3 Correlates with Decreased Macroautophagy Activity—A previous report indicated that Bag3 inhibits rather than stimulates the proteasomal degradation of proteins, suggesting that a alternative mechanism of protein degradation might be stimulated by Bag3 (9Doong H. Rizzo K. Fang S. Kulpa V. Weissman A.M. Kohn E.C. J. Biol. Chem. 2003; 278: 28490-28500Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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Macroautophagy is a bulk degradation process capable of degrading portions of cytoplasm and organelles (32Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3218) Google Scholar, 33Ohsumi Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (1044) Google Scholar). On the basis of these findings we investigated the implication of macroautophagy in the Bag3 mechanism of action. We first asked whether the accumulation of SDS-insoluble HA-Htt43Q observed following HspB8/Bag3 depletion correlated with an altered macroautophagy activity. This question was addressed by measuring the expression of the macroautophagy marker LC3. LC3, a mammalian homologue of yeast Atg8, undergoes complex post-translational modifications leading to 2 main forms: LC3 I, which has a diffuse cytoplasmic distribution and LC3 II, a lipidated form of LC3 I specifically found associated with macroautophagic vacuoles at all stages of formation (18Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar, 34Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Levels of LC3 II are considered a good indicator of macroautophagy activity. Intriguingly, we found that knocking down Bag3 alone or with HspB8, but not knocking down HspB8 alone, produced a decrease in the endogenous level of LC3 II (Fig. 4, black arrowheads). These results strongly suggested that macroautophagy is involved in the HspB8/Bag3 mechanism of action. HspB8/Bag3 Stimulates Macroautophagy—To determine whether the accelerated degradation of Htt43Q observed upon overexpression of HspB8/Bag3 could be due to a stimulation of macroautophagy, we analyzed the effect of HspB8 or Bag3 overexpression on the levels of LC3 II. Myc-LC3 was transfected in HEK-293T cells alone or with either HspB8 or His-Bag3. Western blot analysis of the cell extracts using anti-Myc confirmed the accumulation of LC3 II upon expression of HspB8 or Bag3 (F
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