BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

ABSTRACT

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.

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 β) 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 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 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 BAF-treated cells. We used an additional antibody to detect the reduction in LC3B levels in BAG3 knockdown in HeLa cells (Fig. S1F).

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 (pBAGΔ) 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 <0.05; **, P<0.01 vs. control; ^##, P<0.01; ^###, P<0.001 vs. BAF-treated control.

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 (pBAGΔ),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.

Figure 2.

Effect of BAG3 on lysosomal and proteasomal degradation of LC3B. HeLa cells were transfected with control (siControl) or BAG3 (siBAG3) siRNAs. (A) Transfected cells were treated with NH₄Cl (5 mM) during the last 24 h. Protein levels were analyzed by western blot. Representative images and quantification of total LC3B (LC3B-I plus LC3B-II) normalized by ACTB are shown. (B) Transfected cells were incubated with LysoTracker Green (50 nM) and fluorescence was evaluated by flow cytometry. The average value of fluorescence is shown. (C) Transfected cells were treated with MG132 (2 µM) during the last 24 h. Protein levels were analyzed by western blot. Representative images and quantification of total LC3 (LC3B-I plus LC3B-II) normalized by ACTB are shown. UBB (ubiquitin B) is shown as a control for MG132. (D) siRNA transfected cells were newly transfected after 24 h with GFPµ plasmid. Fluorescence was evaluated by flow cytometry. Percent of fluorescent cells is shown in representative images. The quantification of GFPµ fluorescence is also shown. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical significance was calculated using ANOVA and/or Student t test. **, P < 0.01 vs. siControl; ^###, P < 0.001 vs. NH₄Cl or MG132-treated control.

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 µ (GFPµ) fluorescence, a reporter for proteasomal activity,²⁵ indicating an increased GFPµ degradation (Fig. 2D). These data suggest that BAG3 knockdown increases proteasomal activity. To verify that GFPμ was being degraded by the proteasome, cells were incubated with MG132. As expected, inhibition of the proteasome prevented the decrease in GFPµ 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 protein produced by the overexpression of BAG3 (Fig. 3A). To check that actinomycin D was effectively reducing mRNA transcription, we performed 2 control assays. In the first one, we evaluated if actinomycin D treatment decreased the levels of several mRNAs, including LC3B, BAG3, SQSTM1/p62 (sequestosome 1), HSPA9/mtHSP75 (heat shock 70 kDa protein 9 [mortalin]) and TRAP1/HSP90L (TNF receptor-associated protein 1) (Fig. S5A). In the second assay we used Click Chemistry to evaluate the global RNA transcription using an alkyne-modified nucleoside EU (5-ethynyl uridine) coupled to an azide-derivatized fluorophore (Fig. S5B). To establish whether BAG3 controls the transcription of LC3B, we evaluated the mRNA levels of LC3B (MAP1LC3B) and the family of related genes. Fig. 3B shows that BAG3 overexpression did not changed the mRNA levels of any LC3B-related mRNAs. When BAG3 was knocked down using siBAG3, LC3B (MAP1LC3B) mRNA levels also did not change (Fig. S5C), but an increase about 1.8 fold was observed with MAP1LC3A mRNA. This could be a compensatory response to the lack of MAP1LC3B when BAG3 is knocked down. Together these results show that BAG3 regulates the LC3B protein levels through a mechanism that does neither involve the degradation of LC3B or its transcription.

Figure 3.

Effect of BAG3 on LC3B transcription. (A) HeLa cells were transfected with control (pControl) or a BAG3 (pBAG3) plasmids. Actinomycin D (3 µg/mL) and BAF (10 nM) were added for 6 h and 5 h, respectively, and protein levels were analyzed by western blot. Representative images and quantification of total LC3B (LC3B-I plus LC3B-II) normalized by ACTB are shown. (B) HeLa cells were transfected with control (pControl) or BAG3 (pBAG3) plasmids. Total RNA was isolated at 24 h. LC3B-related family mRNAs were quantified by RT-qPCR. Data are expressed as fold of change relative to pControl and correspond to mean ± SEM of at least 3 independent experiments. Statistical significance was calculated using ANOVA and/or Student ttest. *, P <0.05 vs. respective control; ^#, P<0.05 vs. respective control with actinomycin D; ns: nonsignificant vs. pControl.

BAG3 controls translation of LC3B mRNA

Then, we investigated whether BAG3 controls LC3B mRNA translation. To our knowledge, there are not reports describing the regulation of LC3B translation, either in basal or stress conditions. Cycloheximide treatment impeded the increase of total LC3B protein level induced by BAG3 overexpression. The same effect was observed when BAF was present (Fig. 4A). As controls for the cycloheximide use, we tested whether the cycloheximide treatment effectively decreased the levels of TP53/p53 (tumor protein p53), a short half-life protein (Fig. S6A); and the total protein translation levels using Click chemistry and L-homopropargylglycine (HPG), a methionine alkyne analog (Fig. S6B). Sodium arsenite, which increases the number of stress granules where the translational machinery is sequestered, was also used to inhibit the mRNA translation.²⁶ Like cycloheximide, the treatment with sodium arsenite blocked the increase in total LC3B protein levels induced by BAG3 overexpression (Fig. 4B). As expected, sodium arsenite increased the number of stress granules as detected using an antibody against G3BP1 (GTPase-activating protein [SH3 domain] binding protein 1) (Fig. S6C). Although it has been described that cycloheximide and sodium arsenite can induce cell death, in our conditions, none of the treatments increased necrosis (Fig. S6D) or apoptosis (Fig. S6E). To confirm the effect of BAG3 on LC3B translation, we evaluated the specific translation of LC3B mRNA. A pulse of [³⁵S]methionine/cysteine was used to tag the newly synthesized proteins and then LC3B was immunoprecipitated. BAG3 knockdown decreased the newly synthesized LC3B-I protein (Fig. 4C). Taken together, our results suggest that BAG3 controls LC3B mRNA translation.

Figure 4.

Effect of BAG3 on LC3B translation. HeLa cells were transfected with control (pControl) or BAG3 (pBAG3) 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 (A) cycloheximide (10 µg/mL) and BAF (10 nM) were added during the last 6 h and 5 h, respectively. (B) Sodium arsenite (0.2 mM) and BAF (10 nM) were added during the last 6 h and 5 h, respectively. (C) HeLa cells were transfected with control (siControl) or BAG3 (siBAG3) siRNAs and then maintained in a methionine and cysteine free medium. A [³⁵S]methionine/cysteine pulse was added by 30 min and LC3B was immunoprecipitated from the cell lysate. Representative images of autoradiography and western blot of LC3B immunoprecipitation are shown. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical significance was calculated using ANOVA test. *, P < 0.05 vs. pControl.

LC3B lipidation is independent of the LC3B basal levels controlled by BAG3

Previously, it has been shown that BAG3 participates in selective autophagy.¹³ However, no detailed knowledge about its role in stress-induced autophagy is currently available. Two stress conditions to induce autophagy were chosen to investigate this point, nutrient deprivation and inhibition of proteasome. EBSS (Earle's balanced salt solution) a medium free of amino acids and serum was used to induce autophagy. We found that EBSS treatment increased total protein levels of BAG3, as was described previously¹³ (Fig. 5A). Although BAG3 knockdown reduced LC3B-II total protein levels (normalized by ACTB [βactin]), the ratio LC3B-II to LC3B-I was not modified in EBSS-treated HeLa cells as compared to control cells. These data suggest that the mechanism of LC3B lipidation was not affected by BAG3 knockdown. Next, we evaluated the role of BAG3 on autophagy triggered by the proteasome inhibitor MG132. As shown previously,^18,19 MG132 treatment also increased total protein levels of BAG3 (Fig. 5C). As observed in nutrient deprivation, MG132 treatment reduced LC3B-II absolute levels but not the ratio LC3B-II to LC3B-I (Fig. 5D). Because BAG3 knockdown reduced basal LC3B-II absolute levels, we hypothesized that this reduction could produce a decrease in basal level of autophagy. To test this hypothesis we evaluated total mitochondria levels using MitoTracker Green labeling. As expected BAG3 knockdown increased around 15% the amount of mitochondria (Fig. 5E). These data indicate that BAG3 controls basal autophagy by regulating LC3B total protein level without affecting the LC3B lipidation mechanism.

Figure 5.

Effect of BAG3 on LC3B lipidation in nutrient-deprived and MG132-treated cells. (A) HeLa cells were treated with EBSS (nutrient deprivation medium) by 2 h. (B) HeLa cells were transfected with control (siControl) or BAG3 (siBAG3) siRNAs. BAF (10 nM) was added during the last 5 h and EBSS during the last 2 h. (C) HeLa cells were treated with MG132 (5 µM) by 8 h. (D) HeLa cells were transfected with control (siControl) or BAG3 (siBAG3) siRNAs. MG132 (5 µM) was added during the last 8 h and BAF (10 nM) during the last 5 h. In all figures, protein levels were analyzed by western blot. Representative images and quantification of total LC3B (LC3B-I plus LC3B-II) normalized by ACTB are shown. Data are expressed as mean ± SEM of at least 3 independent experiments. (E) HeLa cells incubated with 200 nM MitoTracker Green for 30 min. After washing, cells were trypsinized and MitoTracker Green fluorescence was determined by flow cytometry. Data are expressed as mean ± SEM of 6 independent experiments. Statistical significance was calculated using ANOVA and Student ttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. Control or siControl; ^#, P < 0.05; ^##, P < 0.01 vs. siBAG3 without treatment.

Discussion

Most information regarding the mechanism of autophagy comes from studies employing stress conditions, mainly nutrient deprivation. Therefore, proteins and mechanisms that control autophagy during stress are well described, while regulation of basal autophagy is mostly unknown. Our results showed that BAG3 regulates LC3B total protein levels, a key autophagic protein, in HeLa and HEK293 cells. The control of LC3B total protein level could be an important mechanism to regulate the fine-tuning of basal autophagy. In HeLa cells, BAG3 modulate LC3B protein levels by controlling its mRNA translation. This effect is apparently unique to LC3B since other ATG proteins were not affected. Furthermore, our results show that BAG3 did not alter the conversion of LC3B-I to LC3B-II by 2 different stress conditions, nutrient deprivation and proteasome inhibition.

Previous reports have shown that BAG3 regulates LC3B-II levels, either by silencing or overexpressing BAG3. However, in these articles, authors do not investigate the mechanism involved on this regulation. Liu et al.¹⁸ show that BAG3 silencing with shRNA reduces the autophagy induced with several proteasome inhibitors in HepG2 cells. Their results show that shRNA BAG3 reduces both LC3B-I and LC3B-II total protein levels, but only the reduction in LC3B-II is analyzed. This study does not take into account the reduction in LC3B-I total protein levels. Moreover, by reviewing the published data we determined that the LC3B-II to LC3B-I ratio was also not affected. The changes in both, LC3B-I and LC3B-II, could be explained by an increase on autophagic flux. However, our experiments with BAF showed that BAG3 did not increase the autophagy flux. Additionally, Liu et al.¹⁸ show that BAG3 silencing does not change LC3B mRNA levels, which is in agreement with our results. Merabova et al.¹⁹ describe that the WW domain of BAG3 is necessary for autophagy induction in human glioblastoma cells. They show that BAG3 overexpression increases both LC3B-II and LC3B-I protein levels. However, they interpret their results only from the standpoint of the LC3B-II increase. Those data highlight the importance of analyzing LC3B-II levels normalized by ACTB, as well as LC3B-II levels normalized by LC3B-I. Discrepancies in the criterion of LC3B normalization are very old and have hindered the proper interpretation of the results on autophagy. The last autophagy guidelines to monitor autophagy suggest showing the results only as LC3B-II normalized by ACTB.²⁴ This recommendation is oriented to reduce errors when an increase in autophagic flux reduces both proteins, LC3B-I and LC3B-II. However, this way of interpreting the results could interfere with information about changes in LC3B expression.

There is little information about the regulation of key autophagy classic checkpoints by BAG3. There is only one description showing that autophagy is controlled by BAG3 through a BECN1 (Beclin 1, autophagy related)-independent mechanism.¹⁸ Moreover, BAG3 expression stabilizes the interaction between BCL2 and BECN1, a complex that prevents class III PtdIns3K activation.¹⁹ This new information does not argue against a role of BECN1 on BAG3-dependent autophagy, but suggests that BAG3 could control the autophagy signaling through a nontraditional mechanism.

LC3B-II degrades with its cargo in the autolysosome^7-9 and LC3B-I is processed and degraded by the proteasome.²³ Therefore, it would be possible that BAG3 regulates the LC3B total protein level by increasing its degradation. However, this is obviously not the case since the incubation with inhibitors of both pathways, MG132 and NH₄Cl, did not prevent the reduction in LC3B total levels induced by BAG3 knockdown, in HeLa cells. We also observed that BAG3 knockdown increased proteasome activity, as previously described.¹³ However, an increase in proteasomal activity was not associated with an enhanced LC3B degradation.

Our results showed that BAG3 does not regulate the LC3B transcription, in HeLa cells. In fact, modulation of BAG3 total protein levels did not change the LC3B mRNA levels and actinomycin D did not affect the total LC3B increase induced by BAG3 overexpression. Moreover, inhibition of translation with sodium arsenite and cycloheximide reversed the increase in LC3B total protein levels induced by BAG3 overexpression. Moreover, LC3B immunoprecipitation after a pulse of [³⁵S]-methionine/cysteine also supports the idea that BAG3 regulates the translation of LC3B mRNA.

BAG3 is a highly dynamic protein that interacts with a multiplicity of effectors, making it difficult to identify the target involved in the translational control of LC3B. A recent study evaluated the entire BAG3 interactome in HeLa cells.²⁷ This work found 382 proteins that interact with this chaperone. Among them were identified transferases, nucleic acid binding proteins, transcription factors, proteases and other chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. Particularly interesting is the description given by Chen et al.²⁷ regarding the idea that BAG3 interacts with some translation initiation factors (as EIF4A2 and EIF4EBP2) and some of ribosomal proteins (RPS12, RPS2, and RPSA). Whether BAG3 directly regulates the activity of these factors to control LC3B translation remains to be elucidated. Another possibility would be to determine whether BAG3 is a ribonucleoprotein that binds directly to LC3B mRNA.

The effect of BAG3 is apparently unique for the translation of LC3B mRNA, because the levels of other ATG proteins did not change upon BAG3 protein level modification. This selectivity argues for a specific BAG3-dependent LC3B mRNA translation regulation, although our results do not exclude the possibility that BAG3 regulates another members of the MAPLC3 family too. Also, it would be important to expand our work to other cell models in order to establish if this mechanism is not only exclusive for HeLa or HEK293 cells.

The mechanism of BAG3 to control the LC3B mRNA translation could involve the regulation of microRNAs or stress granules. Recently, it was shown that BAG3 regulates both positive and negatively several microRNAs. It has been described that BAG3 knockdown decreases the expression of MCL1 (myeloid cell leukemia 1), an antiapoptotic protein, by overexpression of MIR29B.²⁸ Conversely, stress granules are structures wherein the translational machinery is sequestered under stress conditions.²⁹ Stress granules could also explain the selective regulation of mRNA, because specific mRNAs are sequestered with the translational machinery. Our results with sodium arsenite suggest such possibility.

An important point emerging from our data in HeLa cells, is that BAG3 only affects LC3B total protein levels, but does not affect its stress-induced lipidation. These data suggest the possibility that BAG3 could be involved in the regulation of basal autophagy by controlling the amount of LC3B required to remove waste elements produced during basal cellular activity. In fact, our results showed that in BAG3 knockdown HeLa cells, 15% more mitochondria were found, suggesting the occurrence of lower mitochondria degradation. It has previously established that BAG3 is involved in the degradation of proteins by selective autophagy, helping in the selection and transport of target proteins to areas with high autophagic activity.¹⁴ Therefore, BAG3 can sense the amount of protein necessary to be degraded within the cell. As a result, regulation of LC3B total protein levels by BAG3 in HeLa cells, could be considered as a fine tuning of basal autophagy.

Materials and methods

Culture conditions and treatment

HeLa, HEK293 and HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under 5% CO₂. These cells were obtained from American Type Culture Collection (ATCC®; CCL⁻2™, CRL-1573™, CRL-3216™, respectively) and were used for 15 passages. To induce autophagy, cells were treated with Earle's medium balanced salt solution (EBSS; Sigma-Aldrich, E7510) for 2 h or with 5 µM MG132 (Calbiochem-EMD Millipore, 474787) for 8 h. To determine the autophagy flux, cells were treated with 10 nM BAF (Calbiochem-EMD Millipore, 19–148) for 5 h and 30 µM hydroxychloroquine for 4 h (Sigma-Aldrich, H0915). Also, cells were treated with 5 mM NH₄Cl for 24 h. To inhibit gene transcription, the cells were treated with 3 µg/mL actinomycin D (Sigma-Aldrich, A1410) for 6 h. To inhibit translation, cells were treated with 10 µg/mL cycloheximide (Sigma-Aldrich, C7698) for 6 h or 0.2 mM sodium arsenite for 6 h.

Transfection of siRNAs and plasmids

For transient BAG3 knockdown, cells were transfected in Optimem medium (ThermoFisher Scientific, 31985–088) with Oligofectamine (ThermoFisher Scientific, 12252–011) according to the manufacturer's instructions, using 100 nM of the following siRNA: siBAG3 1: 5′-CUGAUGAUCGAAGAGUAUU[dT][dT]-3′; siBAG3 2: 5′-GCAAAGAGGUGGAUUCUAA[dT][dT]-3′. The protein levels were evaluated 48 h after transfection. To induce transient BAG3 overexpression, cells were transfected in Optimem medium with Lipofectamine 2000 (ThermoFisher Scientific, 11668–019) according to the manufacturer's instructions, using 1 µg/mL of the following plasmids: pBAG3, ppxxp-BAG and pBAGΔ. The protein levels were evaluated 24 h or 48 h after transfection.

Production of stable knockdown cells

HEK293T cells were transfected in Optimem medium with Lipofectamine 2000 according to the manufacturer's instructions, using the following plasmids: pMD2VSV.G, pMDLg/pRRE, pRSV-REV and pLKO.1 shRNA against BAG3 (shBAG3 1: 5′GAAGGCAAGAAGACTGACAAA-3′; shBAG3 2: 5′CCTGGACACATCCCAATTCAA-3′). After 48 h, the conditioned medium with lentiviral particles was collected and filtered with 0.45 μm pore filters and added to HeLa cells. After 48 h of viral transduction, HeLa cells shBAG3 were selected in medium containing 2 μg/mL puromycin (Gibco, Life Technologies, A11138–02).

Western blotting

Cells were lysed in lysis buffer (100 mM Tris, 300 mM NaCl, 1% NP40 [AMRESCO, J619], pH 8.0) with protease (Roche Diagnostics, 04693132001). and phosphatases (Roche Diagnostics, 04906845001) inhibitors. Equivalent amounts of protein (20 µg) were separated using 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Blots were probed with the following antibodies: anti-BAG3 (Abcam, 47124), anti-LC3B (Cell Signaling Technology, 2775), anti-ATG12 (Cell Signaling Technology, 2010), anti-ATG7 (Cell Signaling Technology, 8558) and anti-ATG3 (Cell Signaling Technology, 3415), anti-ATG4B (Sigma-Aldrich, A2981), anti-ACTB (Sigma-Aldrich, A5316), anti-UBB (Santa Cruz Biotechnology, sc-8017), anti-TP53 (Santa Cruz Biotechnology, sc-98), anti MTOR (Cell Signaling Technology, 2983), anti phospho-MTOR (Cell Signaling Technology, 2971), anti AMPK (Cell Signaling Technology, 2532) and anti phospho-AMPK (Cell Signaling Technology, 2535).

Immunofluorescence assay

Cells were fixed in paraformaldehyde (4% w/v), permeabilized in Triton X-100 (0.01% w/v; Merck Millipore, 108643) and blocked in BSA (5% w/v; Winkler LTDA, BM-0150). Then they were incubated overnight at 4°C with primary antibody and revealed with Alexa Fluor antibodies (Molecular Probes, Life Technologies, A-31558 and A-21048). Confocal microscopy was performed using a LSM 5 microscope (Zeiss, Jena, Germany) equipped with a 63X objective.

RNA isolation and real-time PCR

Total RNA was extracted from cells with a kit (Macherey–Nagel GmbH & Co, 740955.10). The RNA was used to synthesize cDNA and RT-qPCR was performed on an iCycler PCR Thermocycler (Bio-Rad, Munich, Germany) using the Brilliant II SYBR Green qPCR Master Mix (Agilent Technologies, 600828). A human autophagy primer library masterplate for SYBR green-based Real-Time PCR containing 88 primer sets directed against autophagy genes and 8 housekeeping gene primer sets was used for the analysis of autophagy-related gene expression (Biomol Gmbh, HATPL-I), sequences not commercially available. Amplification of the DNA products was measured in real time using Biorad iQ5 software (Bio-Rad).

Labeling of LC3 with [³⁵S]-methionine/cysteine

Cells were incubated for 60 min in 5 mL of methionine and cysteine-free medium (supplemented with 25 mM HEPES, pH 7.4, 1 mM pyruvate, 2 mM glutamine and 10% fetal bovine serum). 250 μCi of [³⁵S]methionine/cysteine (PerkinElmer Inc., NEG772) was added and cells were incubated for 30 min. Cells were lysed and 400 µg were incubated with 20 µL of agarose-protein G (Santa Cruz Biotechnology, sc-500778) and 2 µg anti-LC3B (kindly donated by Dr. J.A. Hill, University of Texas Southwestern Medical Center). Immunoprecipitates were separated in 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were exposed to CLXposure films (ThermoFisher Scientific, 34089) at least 24 h at −80°C.

Cell survival assays and flow cytometry

Cell viability was determined by measuring incorporation of the vital dye propidium iodide (1 μg/mL), in nonpermeabilized and permeabilized cells. To quantify the lysosomal content, cells were incubated with 50 nM LysoTracker Green (ThermoFisher Scientific, L-7526) for 1 h. The activity of proteasome was performed using transient transfection of GFPμ plasmid. A flow cytometer FACS Canto 1 (Becton Dickinson, NJ, USA) was used for fluorescence quantification.

Click chemistry

RNA transcription and protein translation in cells treated with actinomycin D and cycloheximide, respectively was measured using Click Chemistry following the manufacturer's instructions. Briefly, RNA transcription was assayed using the Click-iT® RNA Alexa Fluor® 488 Imaging Kit (ThermoFisher Scientific, C10329) that employs an alkyne-modified nucleoside EU (5-ethynyl uridine), which is supplied to cells and incorporated into nascent RNA.³⁰ The small size of the alkyne tag enables efficient incorporation by RNA polymerases. Detection of incorporated EU is accomplished by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore, enabling the detection of global RNA transcription. For the analysis of protein translation we used the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit (ThermoFisher Scientific, C10428), which uses L-homopropargylglycine (HPG), a methionine alkyne analog; and Alexa Fluor® 488 azide. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 488 azide leads to a chemoselective ligation or “click” reaction between the green fluorescent azide and the alkyne, allowing the modified proteins to be detected by confocal or fluorescence microscopy.

Mitochondria quantification

HeLa cell mitochondria were labeled with 200 nM MitoTracker Green (ThermoFisher Scientific, M-7514) for 30 min. After washing, cells were analyzed by flow cytometry using a FACScan system (Becton Dickinson, NJ, USA). MitoTracker Green fluorescence intensity was used as a marker of the amount of mitochondria.

Statistical analyses

Data were expressed as the average ± SEM of at least 3 independent experiments. Statistical analyses were carried out with GraphPad Prism software (GraphPad) by means of Student t test analysis or ANOVA. Comparisons between groups were performed with Dunnett or Bonferroni tests for one-way ANOVA or 2-way ANOVA, respectively. Statistical significance was defined as P < 0.05.

Abbreviations

ACTB/β-actin

actin, β

AMPK

AMP-activated protein kinase

ATG

autophagy-related

BAG3

BCL2-associated athanogene 3

BECN1

Beclin 1 autophagy related

EBSS

Earle's balanced salt solution

GFPµ

green fluorescent protein µ

HSP

heat shock protein

MAP1LC3B/LC3B

microtubule-associated protein 1 light chain 3 β

MTOR

mechanistic target of rapamycin (serine/threonine kinase).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We want to thank to Fidel Albornoz and Gindra Latorre for their excellent technical assistance.

Funding

This work was supported by grants from the Comision Nacional de Investigacion Cientifica y Tecnologica de Chile (FONDAP 15130011 to S.L. and M.C.; ACT 1111 to S.L. and M.C.; PhD Grant 24110051 to A.R.). A.R.; C.L.C.; D.P.O. holds a PhD fellowship from Conicyt. A.R. also holds a Beca CHILE for a short research stay 201215442820. The work of C. Behl was supported by the DFG/Collaborative Research Center 1080 (principal investigator CB) and the Corona Foundation.