AUTOPHAGY 2016, VOL. 12, NO. 2, 287–296 http://dx.doi.org/10.1080/15548627.2015.1124225
BASIC RESEARCH PAPER
BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism pez-Crisostoa, Daniel Pen ~ a-Oyarzu na, Daniela Salasa, Valentina Parraa, Clara Quirogaa, Andrea E. Rodrıgueza, Camila Lo c a c Tobias Morawe , Mario Chiong , Christian Behl , and Sergio Lavanderoa,b,d a
Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences & Faculty of Medicine, University of Chile, Santiago, Chile; bCenter for Molecular Studies of the Cell (CEMC), Institute for Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile; c Institute of Pathobiochemistry, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany; dDepartment of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA
ABSTRACT
ARTICLE HISTORY
Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.
Received 13 April 2015 Revised 5 November 2015 Accepted 18 November 2015
Introduction Autophagy is a process that removes vital components of the cell, so its activity must be tightly regulated. The function of the autophagy-related (ATG) proteins is modulated by post-translational modifications such as phosphorylation, glycosylation, acetylation, ubiquitination, proteolysis, and lipidation.1 MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 b) is an ortholog of yeast Atg8, that in mammalian cells includes MAP1LC3 (MAP1LC3A, MAP1LC3B, and MAP1LC3C), GABARAP (GABA[A] receptor-associated protein), GABARAPL1 (GABA[A] receptor-associated protein like 1) and GABARAPL2/ GATE-16 (GABA[A] receptor-associated protein like 2).2 LC3 is cotranslationally processed by ATG4B, a cysteine protease, to generate LC3B-I.3 ATG4B action exposes a glycine at the carboxyl terminus of LC3B-I, which is activated by ATG7 and ATG3 and transferred to phosphatidylethanolamine to produce LC3B-II.4 LC3B-II lies on both surfaces of autophagosome (external and internal) where it serves to elongate the membrane and recruit the cargo. In some scenarios, the induction of autophagy is accompanied by increased mRNA levels of certain autophagy genes such as LC3B, ATG5 or ATG12.5 However, little is known about the mechanisms that controls ATG protein translation. Indeed, there are opposite opinions about the need of protein translation in starvation-induced autophagy.6 During
KEYWORDS
ATG8; autophagy; BAG3; cochaperone; LC3; MAP1LC3B
autophagy, several ATG proteins, including LC3B-II, are eliminated with the cargo,7-9 so it is necessary to replenish its levels for maintaining the autophagic activity. BAG cochaperones, such as BAG3, are nucleotide exchange factors for HSPA8/HSC70 (heat shock 70 kDa protein 8) and other members of the HSP70 family, molecular chaperones that allow proper folding of nascent proteins or, when a protein is irreversibly damaged or in excess, guide it to lysosomal or proteasomal degradation.10 In recent studies, BAG3 was shown to control the selective degradation of misfolded proteins by autophagy, including polyQ-expanded HTT (huntingtin) and mutant SOD1 (superoxide dismutase 1, soluble).11-14 The mechanism involves BAG3 association with dynein to transport misfolded proteins to the aggresomes, facilitating their clearance by autophagy.14 Besides the role in folding and degradation of proteins, recently the role of the HSP70 chaperones in the regulation of the translation of proteins has been described. HSP70 is associated with the nascent polypeptide, assisting in the folding of newly synthesized proteins and acting as sensor for downstream folding conditions.15-17 It is well established that BAG3 increases LC3B lipidation.18,19 However, the mechanism used by BAG3 to increase LC3B-II remains unknown. Here, we described the effects of the manipulation of BAG3 levels on total LC3B protein levels
CONTACT Sergio Lavandero [email protected] Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Olivos 1007, Santiago 8380492, Chile. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis Group, LLC
288
A. E. RODRıGUEZ ET AL.
after siRNA and shRNAknockdown and plasmid overexpression in HeLa cells. We found that BAG3 controls the total levels of LC3B protein by regulating protein translation. BAG3 did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition.
Results BAG3 modulates the total LC3 protein levels without affecting other ATG proteins BAG3 is a protein that positively regulates autophagy.11,13,14,20 BAG3 acts by selectively directing proteins to aggresomes, where autophagy has high activity.14 However, the mechanism that BAG3 uses to activate the autophagy signaling remains unknown. In this study, total protein levels of LC3B (LC3B-I plus LC3B-II) were analyzed in HeLa cells following modulation of the expression of BAG3, to evaluate changes in the expression of LC3B protein. The amount of total LC3B protein decreased significantly when BAG3 is knockdown with 2 different siRNA sequences in HeLa cells (Fig. 1A). This decrease could be explained by an increase in autophagy flow leading to increased LC3B-II degradation. However, in the presence of bafilomycin A1 (BAF), total LC3B protein levels remained low. This decrease in the amount of total cellular LC3B occurred in parallel with the changes in the amount of LC3B-II when BAG3 was knocked down (Figs. S1A and B) or overexpressed (Fig. S1C). This reduction was also observed when compared to nontransfected cells (Fig. S1A). The use of BAF (Fig. S1D) and hydroxychloroquine (Fig. S1E) in HEK293 cells treated with BAG3 siRNA had similar effects to that in HeLa cells. Stable silencing of BAG3 with 2 different sequences of shRNA inserted into the genome of HeLa cells, using lentiviral vectors, also decreased total protein levels of LC3B (Fig. 1B). The reduction in LC3B protein levels was also observed in BAFtreated cells. We used an additional antibody to detect the reduction in LC3B levels in BAG3 knockdown in HeLa cells (Fig. S1F). The opposite effect, an increase in total levels of LC3B, was observed when BAG3 was overexpressed, especially with BAF treatment (Fig. 1C). Indeed, the plasmids previously described to overexpress the proline-rich region and BAG domain of BAG3 (ppxxpBAG) or BAG3 without its BAG domain (pBAGD),14 also increased total LC3B protein levels (Fig. 1D). Apparently for the increase in total LC3B protein levels the BAG domain is not necessary but could be dependent on the proline-rich region present in all plasmids used. BAG3 overexpression increased the autophagosomes detected by immunofluorescence (Fig. S1F). This finding is consistent with previous results.13 Then, we investigated whether the BAG3 effect was exclusive for LC3B or affects other ATG proteins. To this end, we evaluated the amount of the most important proteins involved in the LC3B lipidation reaction. When BAG3 was knocked down or overexpressed no changes in the amount of ATG3, ATG4B, ATG12–ATG5 (the antibody detects both the free and bound form of ATG12 to ATG5)
and ATG7 proteins were observed (Fig. 1E and S2). Conversely, regulation by AMP-activated protein kinase (AMPK) and MTOR (mechanistic target of rapamycin [serine/threonine kinase]) has been widely described in the control of stress-induced autophagy,21,22 but the effects of BAG3 on the activity of these proteins are unknown. Our results showed that there were no significant changes in AMPK and MTOR phosphorylation when BAG3 was knocked down in HeLa cells (Fig. S3A) or overexpressed (Fig. S3B). Therefore, the effect of BAG3 was apparently specific for LC3B protein levels and did not depend on MTOR and AMPK phosphorylation. LC3B degradation through lysosome or proteasome pathways is not affected by BAG3 LC3B is a highly dynamic protein, which is degraded with its cargo by the autolysosome and by the proteasome.23,24 In order to analyze the effect of BAG3 on LC3B degradation by the lysosome, HeLa cells were treated with NH4Cl. This compound increases the pH of the acid organelles and inhibits the lysosomal degradation. LC3B reduction induced by BAG3 knockdown was maintained when cells were treated with NH4Cl (Fig. 2A). The NH4Cl treatment results are consistent with the data obtained with BAF (Fig. 1A), which also blocks lysosomal degradation. Moreover, incubation with LysoTracker Green, a lysosome-specific labeling probe, showed a small reduction in the lysosomal mass of BAG3 knockdown cells (Fig. 2B). This labeling was reduced in NH4Cl¡treated HeLa cells (Fig. S4A), showing that LysoTracker Green is specific to acidic organelles. These data suggest that LC3B degradation by the lysosomal pathway is not affected by BAG3 levels. Additionally, we explored whether the reduction in LC3B total levels by BAG3 knockdown is due to an increase in its proteasomal degradation. The treatment with the proteasome inhibitor MG132 did not revert the reduction in total LC3B protein levels induced by BAG3 knockdown (Fig. 2C). These results suggest that BAG3 knockdown does not reduce the total LC3B protein level by increasing its proteasomal degradation. Conversely, it has been suggested previously that BAG3 influences proteasome activity.13 Therefore, we studied the activity of the proteasome in the BAG3 knockdown HeLa cells. BAG3 knockdown reduces the green fluorescent protein m (GFPm) fluorescence, a reporter for proteasomal activity,25 indicating an increased GFPm degradation (Fig. 2D). These data suggest that BAG3 knockdown increases proteasomal activity. To verify that GFPm was being degraded by the proteasome, cells were incubated with MG132. As expected, inhibition of the proteasome prevented the decrease in GFPm fluorescence (Fig. S4B). LC3B gene transcription is not regulated by BAG3 Following the exclusion of the idea that BAG3 affects LC3 degradation, we investigated whether BAG3 regulates LC3B gene transcription. Transcription inhibition with actinomycin D did not reduce the increase in total levels of LC3B
AUTOPHAGY
289
Figure 1. Effect of BAG3 on LC3B protein levels. Protein extracts from HeLa cells were analyzed by western blot. Representative images and quantification of total LC3 (LC3B-I plus LC3B-II) normalized by ACTB are shown. Where indicated, BAF (10 nM) was added during the last 5 h. (A) Cells were transfected with 2 BAG3 (siBAG3 1 and siBAG3 2) or control (siControl) siRNAs. (B) Stable knockdown generated with 2 lentiviral vectors containing BAG3 (shBAG3 1 and shBAG3 2) or luciferase (shLUC) shRNAs. (C) Cells were transfected with control (pControl) or BAG3 (pBAG3) plasmids. (D) HeLa cells were transfected with control (pControl), BAG3 (pBAG3), proline-rich region and BAG domain of BAG3 (ppxxpBAG) and BAG3 without its BAG domain (pBAGD) plasmids. Protein levels were analyzed by western blot. Representative images and quantification of total LC3B (LC3B-I plus LC3B-II) normalized by ACTB are shown. (E) BAG3 was knocked down or overexpressed and ATG proteins (ATG3, ATG12–ATG5, ATG4B and ATG7) were analyzed. Data are expressed as mean § SEM of at least 3 independent experiments. Statistical significance was calculated using ANOVA and/or Student t test. , P
BASIC RESEARCH PAPER
BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism pez-Crisostoa, Daniel Pen ~ a-Oyarzu na, Daniela Salasa, Valentina Parraa, Clara Quirogaa, Andrea E. Rodrıgueza, Camila Lo c a c Tobias Morawe , Mario Chiong , Christian Behl , and Sergio Lavanderoa,b,d a
Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences & Faculty of Medicine, University of Chile, Santiago, Chile; bCenter for Molecular Studies of the Cell (CEMC), Institute for Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile; c Institute of Pathobiochemistry, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany; dDepartment of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA
ABSTRACT
ARTICLE HISTORY
Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.
Received 13 April 2015 Revised 5 November 2015 Accepted 18 November 2015
Introduction Autophagy is a process that removes vital components of the cell, so its activity must be tightly regulated. The function of the autophagy-related (ATG) proteins is modulated by post-translational modifications such as phosphorylation, glycosylation, acetylation, ubiquitination, proteolysis, and lipidation.1 MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 b) is an ortholog of yeast Atg8, that in mammalian cells includes MAP1LC3 (MAP1LC3A, MAP1LC3B, and MAP1LC3C), GABARAP (GABA[A] receptor-associated protein), GABARAPL1 (GABA[A] receptor-associated protein like 1) and GABARAPL2/ GATE-16 (GABA[A] receptor-associated protein like 2).2 LC3 is cotranslationally processed by ATG4B, a cysteine protease, to generate LC3B-I.3 ATG4B action exposes a glycine at the carboxyl terminus of LC3B-I, which is activated by ATG7 and ATG3 and transferred to phosphatidylethanolamine to produce LC3B-II.4 LC3B-II lies on both surfaces of autophagosome (external and internal) where it serves to elongate the membrane and recruit the cargo. In some scenarios, the induction of autophagy is accompanied by increased mRNA levels of certain autophagy genes such as LC3B, ATG5 or ATG12.5 However, little is known about the mechanisms that controls ATG protein translation. Indeed, there are opposite opinions about the need of protein translation in starvation-induced autophagy.6 During
KEYWORDS
ATG8; autophagy; BAG3; cochaperone; LC3; MAP1LC3B
autophagy, several ATG proteins, including LC3B-II, are eliminated with the cargo,7-9 so it is necessary to replenish its levels for maintaining the autophagic activity. BAG cochaperones, such as BAG3, are nucleotide exchange factors for HSPA8/HSC70 (heat shock 70 kDa protein 8) and other members of the HSP70 family, molecular chaperones that allow proper folding of nascent proteins or, when a protein is irreversibly damaged or in excess, guide it to lysosomal or proteasomal degradation.10 In recent studies, BAG3 was shown to control the selective degradation of misfolded proteins by autophagy, including polyQ-expanded HTT (huntingtin) and mutant SOD1 (superoxide dismutase 1, soluble).11-14 The mechanism involves BAG3 association with dynein to transport misfolded proteins to the aggresomes, facilitating their clearance by autophagy.14 Besides the role in folding and degradation of proteins, recently the role of the HSP70 chaperones in the regulation of the translation of proteins has been described. HSP70 is associated with the nascent polypeptide, assisting in the folding of newly synthesized proteins and acting as sensor for downstream folding conditions.15-17 It is well established that BAG3 increases LC3B lipidation.18,19 However, the mechanism used by BAG3 to increase LC3B-II remains unknown. Here, we described the effects of the manipulation of BAG3 levels on total LC3B protein levels
CONTACT Sergio Lavandero [email protected] Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Olivos 1007, Santiago 8380492, Chile. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis Group, LLC
288
A. E. RODRıGUEZ ET AL.
after siRNA and shRNAknockdown and plasmid overexpression in HeLa cells. We found that BAG3 controls the total levels of LC3B protein by regulating protein translation. BAG3 did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition.
Results BAG3 modulates the total LC3 protein levels without affecting other ATG proteins BAG3 is a protein that positively regulates autophagy.11,13,14,20 BAG3 acts by selectively directing proteins to aggresomes, where autophagy has high activity.14 However, the mechanism that BAG3 uses to activate the autophagy signaling remains unknown. In this study, total protein levels of LC3B (LC3B-I plus LC3B-II) were analyzed in HeLa cells following modulation of the expression of BAG3, to evaluate changes in the expression of LC3B protein. The amount of total LC3B protein decreased significantly when BAG3 is knockdown with 2 different siRNA sequences in HeLa cells (Fig. 1A). This decrease could be explained by an increase in autophagy flow leading to increased LC3B-II degradation. However, in the presence of bafilomycin A1 (BAF), total LC3B protein levels remained low. This decrease in the amount of total cellular LC3B occurred in parallel with the changes in the amount of LC3B-II when BAG3 was knocked down (Figs. S1A and B) or overexpressed (Fig. S1C). This reduction was also observed when compared to nontransfected cells (Fig. S1A). The use of BAF (Fig. S1D) and hydroxychloroquine (Fig. S1E) in HEK293 cells treated with BAG3 siRNA had similar effects to that in HeLa cells. Stable silencing of BAG3 with 2 different sequences of shRNA inserted into the genome of HeLa cells, using lentiviral vectors, also decreased total protein levels of LC3B (Fig. 1B). The reduction in LC3B protein levels was also observed in BAFtreated cells. We used an additional antibody to detect the reduction in LC3B levels in BAG3 knockdown in HeLa cells (Fig. S1F). The opposite effect, an increase in total levels of LC3B, was observed when BAG3 was overexpressed, especially with BAF treatment (Fig. 1C). Indeed, the plasmids previously described to overexpress the proline-rich region and BAG domain of BAG3 (ppxxpBAG) or BAG3 without its BAG domain (pBAGD),14 also increased total LC3B protein levels (Fig. 1D). Apparently for the increase in total LC3B protein levels the BAG domain is not necessary but could be dependent on the proline-rich region present in all plasmids used. BAG3 overexpression increased the autophagosomes detected by immunofluorescence (Fig. S1F). This finding is consistent with previous results.13 Then, we investigated whether the BAG3 effect was exclusive for LC3B or affects other ATG proteins. To this end, we evaluated the amount of the most important proteins involved in the LC3B lipidation reaction. When BAG3 was knocked down or overexpressed no changes in the amount of ATG3, ATG4B, ATG12–ATG5 (the antibody detects both the free and bound form of ATG12 to ATG5)
and ATG7 proteins were observed (Fig. 1E and S2). Conversely, regulation by AMP-activated protein kinase (AMPK) and MTOR (mechanistic target of rapamycin [serine/threonine kinase]) has been widely described in the control of stress-induced autophagy,21,22 but the effects of BAG3 on the activity of these proteins are unknown. Our results showed that there were no significant changes in AMPK and MTOR phosphorylation when BAG3 was knocked down in HeLa cells (Fig. S3A) or overexpressed (Fig. S3B). Therefore, the effect of BAG3 was apparently specific for LC3B protein levels and did not depend on MTOR and AMPK phosphorylation. LC3B degradation through lysosome or proteasome pathways is not affected by BAG3 LC3B is a highly dynamic protein, which is degraded with its cargo by the autolysosome and by the proteasome.23,24 In order to analyze the effect of BAG3 on LC3B degradation by the lysosome, HeLa cells were treated with NH4Cl. This compound increases the pH of the acid organelles and inhibits the lysosomal degradation. LC3B reduction induced by BAG3 knockdown was maintained when cells were treated with NH4Cl (Fig. 2A). The NH4Cl treatment results are consistent with the data obtained with BAF (Fig. 1A), which also blocks lysosomal degradation. Moreover, incubation with LysoTracker Green, a lysosome-specific labeling probe, showed a small reduction in the lysosomal mass of BAG3 knockdown cells (Fig. 2B). This labeling was reduced in NH4Cl¡treated HeLa cells (Fig. S4A), showing that LysoTracker Green is specific to acidic organelles. These data suggest that LC3B degradation by the lysosomal pathway is not affected by BAG3 levels. Additionally, we explored whether the reduction in LC3B total levels by BAG3 knockdown is due to an increase in its proteasomal degradation. The treatment with the proteasome inhibitor MG132 did not revert the reduction in total LC3B protein levels induced by BAG3 knockdown (Fig. 2C). These results suggest that BAG3 knockdown does not reduce the total LC3B protein level by increasing its proteasomal degradation. Conversely, it has been suggested previously that BAG3 influences proteasome activity.13 Therefore, we studied the activity of the proteasome in the BAG3 knockdown HeLa cells. BAG3 knockdown reduces the green fluorescent protein m (GFPm) fluorescence, a reporter for proteasomal activity,25 indicating an increased GFPm degradation (Fig. 2D). These data suggest that BAG3 knockdown increases proteasomal activity. To verify that GFPm was being degraded by the proteasome, cells were incubated with MG132. As expected, inhibition of the proteasome prevented the decrease in GFPm fluorescence (Fig. S4B). LC3B gene transcription is not regulated by BAG3 Following the exclusion of the idea that BAG3 affects LC3 degradation, we investigated whether BAG3 regulates LC3B gene transcription. Transcription inhibition with actinomycin D did not reduce the increase in total levels of LC3B
AUTOPHAGY
289
Figure 1. Effect of BAG3 on LC3B protein levels. Protein extracts from HeLa cells were analyzed by western blot. Representative images and quantification of total LC3 (LC3B-I plus LC3B-II) normalized by ACTB are shown. Where indicated, BAF (10 nM) was added during the last 5 h. (A) Cells were transfected with 2 BAG3 (siBAG3 1 and siBAG3 2) or control (siControl) siRNAs. (B) Stable knockdown generated with 2 lentiviral vectors containing BAG3 (shBAG3 1 and shBAG3 2) or luciferase (shLUC) shRNAs. (C) Cells were transfected with control (pControl) or BAG3 (pBAG3) plasmids. (D) HeLa cells were transfected with control (pControl), BAG3 (pBAG3), proline-rich region and BAG domain of BAG3 (ppxxpBAG) and BAG3 without its BAG domain (pBAGD) plasmids. Protein levels were analyzed by western blot. Representative images and quantification of total LC3B (LC3B-I plus LC3B-II) normalized by ACTB are shown. (E) BAG3 was knocked down or overexpressed and ATG proteins (ATG3, ATG12–ATG5, ATG4B and ATG7) were analyzed. Data are expressed as mean § SEM of at least 3 independent experiments. Statistical significance was calculated using ANOVA and/or Student t test. , P
