aa 2 212  (R&D Systems)

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, BT-549 and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Western Blot, Transfection

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. HeLa ARTS knockdown (ARTS KD) cells and Sept4/ARTS KO MEFs show significantly higher levels of steady-state Bcl-2 protein when compared with WT cells. This suggests that ARTS plays an important role in regulating Bcl-2 levels. B. WT and ARTS KD HeLa cells were treated with 1.75 µM STS. Western Blot analyses demonstrate that while decreased Bcl-2 levels were seen in apoptotic WT HeLa cells, Bcl-2 levels in ARTS KD HeLa cells remained unchanged. C. Western Blot analyses of cytosolic fractions of BT-549, HeLa WT and HeLa ARTS KD cells reveal that endogenous Bcl-2 is found in the cytosol of WT STS-treated cells. In contrast, a strong inhibition in translocation of Bcl-2 to the cytosol was seen in ARTS KD HeLa cells. This suggests that ARTS is required for the proper translocation of Bcl-2 from mitochondria to the cytosol upon apoptotic induction. D. Immuno-fluorescence (IF) was performed on HeLa and a stable Bcl-2 knockdown (Bcl-2 KD) cells. The fraction of cells with cytosolic staining of ARTS is represented in the bar chart. While only a small portion of WT untreated (NT) HeLa cells show the presence of ARTS in the cytosol, a significant increase in cells containing cytosolic ARTS was seen following STS treatment. In contrast, the majority of HeLa Bcl-2 KD NT cells exhibit cytosolic ARTS (four fold higher than WT HeLa cells), and, only a slight increase in cells with cytosolic ARTS is seen after STS treatment. (* * p-value ≤ 0.01). See also supplemental Figure S1. E. Cytosolic and mitochondrial fractions of WT MEFs and Bcl-2 KO MEFs were analyzed by WB analysis with COX IV as a mitochondrial and GAPDH as a cytosolic marker. In Bcl-2 KO MEFs, the majority of ARTS was in the cytosol. This suggests that Bcl-2 is involved in localizing ARTS to mitochondria. F. IF of WT MEFs and Sept4/ARTS KO MEFs transiently transfected with GFP-Bcl-2. Cellular localization of Bcl-2 was quantified and the fraction of cells with cytosolic Bcl-2 is shown in the bar charts. While a significant increase in cytosolic Bcl-2 was seen in apoptotic WT MEFs, the levels of cytosolic Bcl-2 in Sept4/ARTS KO MEFs remained unchanged. See also supplemental Figure S1. G. Subcellular fractionation of HeLa cells was followed by in vivo ubiquitylation of each fraction. IgG represents the control cells incubated with non-specific IgG. Poly-ubiquitylated forms of Bcl-2 were seen only in the cytosolic fraction. * Represents the IG heavy chain. This indicates that ubiquitylation of Bcl-2 occurs in the cytosol and that ARTS is required for translocation of Bcl-2 to the cytosol during apoptosis.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Western Blot, Inhibition, Translocation Assay, Fluorescence, Staining, Marker, Transfection, Fractionation, In Vivo, Incubation

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. IP of ARTS in COS-7 cells transiently transfected with 6-Myc-ARTS using a monoclonal anti-ARTS antibody (Sigma-Aldrich). Endogenous ARTS was immunoprecipitated from HeLa and BT-549 cells. Western blot analyses show that in COS-7, HeLa and BT-549 cells, Bcl-2 and XIAP co- precipitate with ARTS. AII. HeLa cells were treated with 1.75 µM STS and IP of ARTS was performed as described in AI. While binding of ARTS to Bcl-2 was seen in non-treated cells (NT), binding of ARTS to XIAP and Bcl-2 is increased in STS treated cells. B. Recombinant His-ARTS, Bcl-2 and GST-XIAP were incubated overnight at 4 °C. Pulldown of GST-XIAP shows that binding of Bcl-2 to XIAP depends on ARTS. C. To assess proximity of proteins, consistent with complex formation, we used BiFC. HeLa cells were transiently transfected with ARTS, Bcl-2 and XIAP fused to parts of YFP YFP-Venus. Jun and bFos, known to form heterodimers, served as a positive control (p.c.). Jun and bFosdelZIP lacking the carboxyl-terminal half of the bFos were used as a negative control (n.c.). The fluorescent signal indicating the proximity of each pair of proteins was measured by flow cytometry. Mean fluorescence intensity (MFI). FACS results were normalized to the readings of transfection efficiency reporter (pdsRED). The values represent mean values ± SE of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01). The Y-axis represents the ratio between YFP fluorescence (reflecting binding of a pair of proteins) and the red fluorescence (marking the transfected cells). FACS analyses reveal that ARTS can bind to both Bcl-2 and to XIAP. Yet, only background levels of fluorescence were seen with XIAP and Bcl2, suggesting that these two proteins do not bind each other. These results indicate that ARTS, XIAP and Bcl-2 form a ternary complex, and that ARTS is required for the formation of this complex. See also supplemental Figure S2.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Incubation, Positive Control, Negative Control, Flow Cytometry, Fluorescence

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. A custom designed Bcl-2 derived peptide array (CelluSpot™) was incubated with recombinant (His)6-Lipo-TEV (HLT-) tagged ARTS. The peptide array was also incubated with recombinant HLT-tag alone which served as a negative control. The arrays were probed with the indicated antibodies. Lower panel depicts the Bcl-2 peptides which bind to ARTS. These peptides are schematically represented across the Bcl-2 domains; the black lines indicate strong binding and the grey lines a weak to moderate binding. B. Bcl-2 expression vectors; full length and four constructs deleting each BH domain in Bcl-2 are illustrated at the top (FLD- Flexible Loop Domain, TM-Transmembrane domain). These vectors were co-transfected with 6myc-ARTS into Bcl-2 KO MEFs. IP of ARTS was performed in MEFs transfected with Bcl-2-BH deletions, WT Bcl-2 (positive control) and empty vector (negative control). A significant reduction in binding of ARTS to Bcl-2delBH3 was seen. See also supplemental Tables S1 and S2. C. BIFC assays were performed as described in Figure 3C. HeLa cells were co-transfected with vectors expressing ARTS, Bcl-2 and Bcl-2delBH3 fused to either VN or VC parts of the YFP Venus fragment (ARTS-VN, Bcl-2-VC. Bcl-2delBH3 –VC). A 47% decrease in the proximity between ARTS and Bcl-2delBH3 was seen compared to WT Bcl-2. Jun/bFos served as positive control (p.c.), and Jun/bFosdel ZIP as negative control (n.c.). Results are shown as mean + S.E. of three independent experiments. (*, p ≤ 0.05; **, p ≤ 0.01). See also supplemental Figure S3. D. In vivo ubiquitylation of Bcl-2 WT and Bcl-2delBH3. MEFs Bcl-2 KO cells were transfected with Flag-Bcl-2WT or Flag-Bcl-2delBH3. Cells were treated with MG132 and STS for one hour or left untreated. Proteins were immunoprecipitated with anti-ubiquitin. Poly-ubiquitylated forms of Bcl-2 were detected using an anti-Bcl-2 antibody. While WT Bcl-2 undergoes ubiquitylation following treatment with STS, no ubiquitylation of Bcl-2delBH3 was seen. See also supplemental Figure S4.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Derivative Assay, Peptide Microarray, Incubation, Recombinant, Negative Control, Binding Assay, Expressing, Construct, Transfection, Positive Control, Plasmid Preparation, In Vivo, Immunoprecipitation

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: AI. HeLa cells were transiently transfected with Bcl-2 or co-transfected with Bcl-2 and XIAP or with Bcl-2 and XIAPdelRing. AII. MEFs were produced from WT mice and from XIAPdelRing 14-day old mouse embryos. WB and densitometry analyses reveal a significant accumulation of Bcl-2 in HeLa cells co-transfected with XIAPdelRing and in XIAPdelRing MEFs. B. Sept4/ARTS KO MEFs were transfected with WT ARTS and a mutant version of ARTS lacking its unique C-terminus (Cdel-ARTS) which cannot bind to XIAP. Sept4/ARTS KO MEFs transfected with WTARTS exhibit decreased levels of Bcl-2 following apoptotic induction (UV), while MEFs transfected with the Cdel-ARTS have increased levels of Bcl-2. C. In vitro ubiquitylation assays were performed by incubating recombinant Bcl-2 with recombinant XIAP, E1, E2-UbcH5b and ubiquitin. In the control reactions the indicated components were excluded. Ubiquitylation of Bcl-2 was seen only upon addition of XIAP. D. In vitro ubiquitylation assays with purified Bcl-2 and recombinant XIAP, cIAP1, Parkin, or Siah2. Only addition of XIAP resulted in the ubiquitylation of Bcl-2. See also Supplemental Figure S5 E. In vivo ubiquitylation in WT and XIAPdelRing MEFs. MEFs were co-transfected with ARTS, Bcl-2 and HA-ubiquitin. Cells were treated with 20 µM MG-132 for 6h and concomitantly treated with 1.75 µM STS and IP was performed with an anti-Bcl-2 antibody. *Represents the heavy chain of the antibody. While significant ubiquitylation of Bcl-2 occurred after treatment with STS in WT MEFs, no ubiquitylation of Bcl-2 was seen in XIAPdelRing MEFs. F. In vivo ubiquitylation assays using WT and XIAP KO MEFs. No ubiquitylation of Bcl-2 was seen in XIAP KO MEFs. Collectively these results show that XIAP serves as the specific E3-ligase for Bcl-2 and is required for its degradation.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Transfection, Produced, Mutagenesis, In Vitro, Recombinant, Purification, In Vivo

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. In vitro ubiquitylation assays were performed by incubating recombinant Bcl-2 with XIAP and ARTS, E1, E2-UbcH5b and ubiquitin in the presence of ATP at 37 °C for 1 hr. A significant increase in the appearance of ubiquitylated forms of Bcl-2 was seen in the presence of ARTS. B. HeLa WT or HeLa ARTS KD cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and STS for 0.5 h. Bcl-2-ubiquitin conjugates were seen in the apoptotic WT HeLa cells, but not in ARTS KD HeLa cells. C. In vivo ubiquitylation assays of Sept4/ARTS KO MEFs transfected with either empty vector or ARTS expression vector, followed by treatment with MG132 and STS for 3 h. *Represents the IgG heavy chain. While no ubiquitylation of Bcl-2 was observed in the Sept4/ARTS KO MEFs, transfection of ARTS restored their ability to generate poly-ubiquitylated forms of Bcl-2 upon induction of apoptosis.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: In Vitro, Recombinant, Transfection, In Vivo, Plasmid Preparation, Expressing

Journal: Cell reports

Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

doi: 10.1016/j.celrep.2017.09.052

Figure Lengend Snippet: A. Schematic representation of Bcl-2 highlighting its four lysine residues. B. MS/MS spectrum spanning the ubiquitylation site in Bcl-2. The trypsin cleaved peptides were enriched for Gly-Gly peptides using Pilot Ubiquitin Remnant Motif (K-ε-GG) kit and subjected to liquid chromatography-mass spectrometry separation. Precursor ion (brown) represents the uncleaved GFP-Bcl-2. Lines represent the relative abundance of detected peptides. The lines in black represent peptides that are not cleaved products of the precursor ion (y1–y6) and precursor ion of the un-fragmented peptide (brown). Lines in green (y1–y8) represent the cleaved GFP-Bcl-2 peptides and their corresponding amino acid sequence. KGG (red) represents an ubiquitylated lysine. This analysis identified Lysine 17 in Bcl-2 as the acceptor for XIAP-mediated ubiquitylation. C. MEFs Bcl-2 KO and HeLa cells were transfected with expression vectors for WT Bcl-2 (WT), Bcl-2 containing a substitution mutation of Lysine 17 into Alanine (K17A), and Bcl-2 in which all Lysines were changed to Alanine (No K). Increased levels of mutant Bcl-2 (K17A, No K) were seen upon apoptotic induction. This was accompanied by a decrease in apoptosis, as shown with three different apoptotic markers. Densitometry analyses are shown in the lower panel. D. Bcl-2 KO MEFs and HeLa cells were transfected with Bcl-2 as in (C) together with a GFP-cleaved caspase-3 reporter. Bcl-2 KO MEFs and HeLa cells expressing lysine mutants had significantly less cleaved caspase 3-positive cells compared to WT Bcl-2. These results suggest that Lysine 17 is the main acceptor for ubiquitylation of Bcl-2.

Article Snippet: The array was screened for binding following incubation with the recombinant HLT-ARTS protein and Bcl-2 protein minus C- terminus (827-BC-050, R&D systems).

Techniques: Tandem Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Sequencing, Transfection, Expressing, Mutagenesis