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. 2007 May 16;26(10):2527-39.
doi: 10.1038/sj.emboj.7601689. Epub 2007 Apr 19.

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

M Chiara Maiuri  1 , Gaëtane Le Toumelin, Alfredo Criollo, Jean-Christophe Rain, Fabien Gautier, Philippe Juin, Ezgi Tasdemir, Gérard Pierron, Kostoula Troulinaki, Nektarios Tavernarakis, John A Hickman, Olivier Geneste, Guido Kroemer
Affiliations

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

V体育ios版 - M Chiara Maiuri et al. EMBO J. .

"V体育官网入口" Abstract

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy VSports手机版. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L). .

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Figures

Figure 1
Figure 1
Physical interaction between Bcl-XL/Bcl-2 and the BH3-like domain of Beclin-1. (A) Selection of Beclin-1 fragments that interact with Bcl-XL/Bcl-2 in the yeast-two-hybrid system. Black lines indicate the fragments of Beclin-1 (grey) that interact with Bcl-XL (upper part of the graph) or with Bcl-2 (lower part). ▪, CEMC7 library; •, human thymocyte library; ▴, placenta library and ★, human brain library. The minimal interacting domain required for interaction with Bcl-XL/Bcl-2 is situated between aa 112 and 159. (B) Alignment of the BH3-like domain of Beclin-1 with established BH3 domains. L116A Beclin-1 and F123A Beclin-1 describe two Beclin-1 mutant peptides. (C) Fluorescence polarization assays demonstrating that a peptide containing the BH3-like domain of Beclin-1 interacts with Bcl-XL. Recombinant Bcl-XL ΔTM was incubated with carboxyfluorescein-labeled Bak BH3 peptide, in the absence or presence of wild-type (WT) Beclin-1 BH3 or either of the two mutant peptides described in B. (D) Affinity determination of the interaction between recombinant Bcl-XL ΔTM and fluorescent Beclin-1 BH3, by fluorescence polarization. A comparison between WT Bcl-XL(squares) and G138A Bcl-XL (triangles) is shown (means±s.d., n=3 separate experiments). (E) Co-immunoprecipitation of Bcl-XL and WT or mutant Beclin-1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag-tagged Bcl-XL and immunochemical detection of Beclin-1. (F) Co-immunoprecipitation of Beclin-1 with-WT and mutant (G138A) Bcl-XL. Results are representative of at least three experiments yielding similar results.
Figure 2
Figure 2
Inhibition of the interaction between Beclin-1 and anti-apoptotic Bcl-2 proteins by ABT737. (A) Competition between Beclin-1 BH3 and ABT737 for Bcl-XL binding. A fluorescent 25-mer peptide containing the BH3-like domain of Beclin-1 (Figure 1D) was docked to recombinant Bcl-XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl-XL ΔTM, was 1.7 μM. (B, C) Abolition of the interaction between Bcl-XL and Beclin-1 by ABT737 in intact cells. Co-immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co-transfected Bcl-XL and Beclin-1 (B) and for endogenous Beclin-1 interacting with Flag-tagged Bcl-XL (C). (D, E) Differential effect of ABT737 on the interaction between Bcl-2 (D) or Mcl-1 (E) and Beclin-1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.
Figure 3abcd
Figure 3abcd
Beclin-1-dependent autophagy stimulated by ABT737. (A, B) Detection of autophagic vacuoles by LC3-GFP and their modulation by ABT737 and by Beclin-1-specific siRNAs. HeLa cells were transfected with control or Beclin-1-specific siRNAs, 24 h later re-transfected with LC3-GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient-free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3-GFP-transfected cells bearing LC3-GFP aggregates in the cytoplasm (LC3-GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin-1-specific siRNAs, as quantified by immunoblot. (C, D) Detection of cytoplasmic vacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin-1-specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmic vacuole (arrow head) (means±s.d., n=3 separate experiments).
Figure 3ef
Figure 3ef
(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown. (F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm-sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments.
Figure 4
Figure 4
Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin-1 at 0 h) were re-transfected with LC3-GFP finally cultured in nutrient-free (NF) conditions (60–72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopic detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A). The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).
Figure 5
Figure 5
Impact of the BH3-like domain on Beclin-1-induced autophagy. (A–D) Modulation of Beclin-1-induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP-LC3 together with an empty control vector or Beclin-1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3-GFP-positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3-GFP method. (E) BH3-dependent modulation of Beclin-1-induced autophagy by anti-apoptotic Bcl-2 proteins. Cells were transfected simultaneously with Beclin-1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl-XL, Bcl-XL G138A, Bcl-2 or Mcl-1 (or control vector), as well as GFP-LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h-long experiment. The percentage of cells exhibiting the accumulation of LC3-GFP in vacuoles (LC3-GFPvac) is quantified as means±s.d. (n=3 separate experiments).
Figure 6
Figure 6
Interaction between endogenous and spatially restricted Bcl-2 and Beclin-1 in starvation and after addition of ABT737. (A) Immunoprecipitation of endogenous Bcl-2 and Beclin-1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient-free EBSS or with ABT737 (1 μM). (B) Immunoprecipitation of wild-type and ER-targeted Bcl-2 with Beclin-1. Cells stably transfected with wild type, ER- and mitochondrion-targeted Bcl-2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. (C, D) Immunoprecipitation of Bcl-2 and Beclin-1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.
Figure 7
Figure 7
Impact of the BH3-only protein Bad on autophagy. (A, B) Interactions between Bcl-XL, Beclin-1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L-690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl-XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl-XL, Beclin-1 or Bad. (C) Interaction between endogenous Bad and Bcl-2. HeLa cells were treated with ABT737 (1 μM) or nutrient-depleted, followed by immunoprecipitation of Bcl-2 and immunodetection of Bad. (D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3-GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin-1-associated PI3 kinase Vps34. Forty-eight hours later, when the siRNAs had down-regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3-GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P<0.05, n=3 separate experiments). (F) Effect of the Bad knock-out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3-GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P<0.05) effect of Bad deficiency. (G) Effect of the Bad knockout on the Beclin-1/Bcl-2 interaction. WT or Bad−/− MEF were subjected to the indicated treatments, followed by immunoprecipitation of Bcl-2 and immunobloting of Beclin-1. (H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3-GFP alone (Control), together with vector-only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan-caspase inhibitor Z-VAD-fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P<0.01). Immunoblots are representative of at least three independent experiments.
Figure 8
Figure 8
The BH3-domain protein EGL-1 modulates starvation-induced autophagy in C. elegans. L4 Larvae expressing an LGG-1∷DsRED reporter (whose expression level provides an indication of autophagy) as well as a gain-of-function mutation of egl-1 (n487) or a deletion allele of egl-1 (ok1418) were maintained in rich medium or starved overnight. (A) Representative images of wt and egl-1 mutant embryos, bearing the plgg−1DsRED∷LGG-1 reporter transgene are shown under conditions of normal growth and under starvation. Scale bars denote approximately 50 μm. (B) Quantification of LGG-1∷DsRED fluorescence as determined in (A). Each point represents the measurement for one individual embryo. Horizontal bars denote means and gray areas denote standard errors.
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