Journal: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: Possible role of NF- κ B and NF- κ B-mediated pro-survival signals in TP/LPS-induced hepatotoxicity. (A) Experimental design to detect the time-dependent changes of NF- κ B and its related pro-survival factors. (B)–(G) Relative mRNA levels of NF- κ B target genes, including Ciap1 , Ciap2 , Flip L , Xiap , Tnfaip3 , and Nfkbia , were detected by qPCR with tubulin as the internal control ( n = 6). (H)–(L) Representative Western blots and relative intensity of protein bands of FLIP L , CIAP1, XIAP, and I κ B- α after the treatment of TP and LPS with tubulin as the loading control ( n = 4–6). (M) Representative photomicrographs of liver sections by IHC for P65 1 h after LPS application (200 × ). Scale bar = 50 μm. Results were expressed as mean ± SEM and statistical analysis was performed using Two-way ANOVA following by Tukey's multiple comparison test. *, # , $ P <0.05, **, ## , $$ P <0.01, ***, ### , $$$ P <0.001; ns, no statistical difference.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Control, Western Blot, Comparison

Journal: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: The primer sequences used for qPCR assay in mice.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 stabilizes and stimulates E2F1 in an E3-ubiquitin ligase activity-dependent manner. ( a, b and d ) E2F1 activity and expression in Hela cells ( a and b ) or in cIAP1 −/− /cIAP2 −/− MEFs ( d ) transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-3HA-E2F1, along with empty vector (Vector), cIAP1, cIAP1-H588A (devoid of E3-ubiquitin ligase activity) or cIAP1-F616A (lacking dimerization capacity) mutant encoding vector. HeLa cells were treated for 24 h with PYR-41 10–35 μ M before analysis ( a ). Upper panels: E2F1 transcriptional activity was assessed in gene luciferase experiments. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1 alone. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t -test. *** P <0.001, **0.001< P <0.01, *0.01< P <0.1, NS P >0.1. Lower panels: the expression of the constructs was analyzed by a western blot analysis. β -Actin or HSC70 are used as loading control. *Unspecific bands. ( c ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1, His-Ubiquitin encoding vectors, with an empty vector, cIAP1 or cIAP1 F616A (F/A) mutant constructs. Ubiquitinated proteins were pulled-down by using non-selective or K63-TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody. ( e ) Western blot analysis of cIAP1 or 3HA-E2F1 expression in HeLa cells transfected with pCMV-3HA-E2F1 and with empty vector (Vector), cIAP1 or cIAP1-F616A mutant (dimerization defective mutant) encoding vector, treated or not with MG132 5 μ M overnight. ( f ) In vitro ubiquitination assay of GST-E2F1 fusion protein immobilized on gluthatione sepharose and incubated with ubiquitin, E1 and E2 recombinant proteins with or without recombinant cIAP1

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Western Blot, Control, In Vitro, Incubation, Recombinant

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 overexpression revealed a K63 ubiquitination of E2F1 on lysine residue 161/164. ( a ) Schematic representation of E2F1 protein structure indicating the lysine residues and lysine clusters. ( a ) Cyclin A: CDK2-binding domain; DBD: DNA-binding domain, dimer: dimerization domain; MB: marked box: Rb: Rb-binding domain. ( b ) Gene luciferase experiments performed in HeLa cells transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-HA (vector), HA-tagged-E2F1 (one single or 3HA) or HA-E2F1 mutants in which indicated K have been mutated into R along with empty vector (vector) or cIAP1-encoding vector. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t test. *** P <0.001, *0.01< P <0.1, NS P >0.1. The expression of the E2F1 constructs was checked by a western blot analysis (upper panel). β -Actin is used as loading control. ( c and d ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1 wt, K137R mutant or K161/164R mutant, His-Ubiquitin, with an empty vector or cIAP1 or cIAP1 F616A (F/A) mutant constructs. E2F1 is IP by using specific anti-E2F1 antibody and ubiquitination is revealed by using pan-ubiquitin antibody (FK2) ( c ), or ubiquitinated proteins were pulled-down by using K63-specific TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody ( d ). ( e ) IP analysis of the interaction of cIAP1 with E2F1 wt or K161/164 R mutant. Hela cells were transfected with 3HA-E2F1 encoding constructs and cIAP1. cIAP1 or E2F1 were IP using anti-cIAP1 or anti-HA antibody and cIAP1-E2F1 interaction was revealed by a western blot analysis. KO: cell lysate from cIAP1 −/− KO MEFs was used to check nonspecific reactivity of the antibody

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Over Expression, Ubiquitin Proteomics, Residue, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Western Blot, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 is required for DNA damage-induced stabilization of E2F1. ( a ) Western blot analysis of E2F1 and cIAP1 in U2OS cells transfected with control or cIAP1-siRNA and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells transfected with cIAP1 siRNA or cIAP1-encoding construct and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (input). β -Actin was used as loading control. ( c ) Ubiquitination assay performed in HeLa cells transfected with control or cIAP1 siRNA and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. Ubiquitinated proteins were pulled-down by using K63 specific TUBEs and ubiquitinated E2F1 is revealed by using E2F1 antibody. ( d ) Western blot analysis of E2F1 in U2OS cells transfected with 3HA-E2F1 or 3HA-E2F1-K161/164R mutant and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( e ) Ubiquitination of E2F1 in HeLa cells transfected with pCMV-3HA-E2F1 or pCMV-3HA-E2F1 K161/164R, and with His-tagged ubiquitin wt, and then treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both untreated samples. ( f ) U2OS cells were transfected with cIAP1 siRNA or treated with 17 nM GDC-0152 for 1 h, and then treated with 10 μ M etoposide for 48 h. tp73 mRNA expression was measured by RT-qPCR. UT: untreated cells. Results were normalized to hprt mRNA and expressed relatively to control untreated cells. Mean±S.D. of three independent experiments

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Western Blot, Transfection, Control, Concentration Assay, Ubiquitin Proteomics, Construct, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: PRMT-mediated arginine methylation of E2F1 is required for regulation of E2F1 by cIAP1. ( a ) Western blot analysis of E2F1 and cIAP1 in HeLa cells transfected with 3HA-E2F1 and cIAP1-encoding constructs, and treated for 24 h with 100 μ M PRMT inhibitor AMI-1. HSC70 was used as loading control. ( b ) Ubiquitination of E2F1 in HeLa transfected with pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt, and then treated for 16 h with 50 μ M AMI-1±10 μ M etoposide for 6 h. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene is checked by western blot. β -Actin was used as loading control. ( c ) E2F1 transcriptional activity as measured by using a gene reporter luciferase assay performed in Hela cells transfected as in a in the presence of CCNE promoter-Firefly luciferase reporter plasmid and treated with increasing concentration of AMI-1 for 24 h. Results are expressed as average±S.D. from at least three experiments. Statistical analysis performed using Student’s t- test. *** P <0.001, **0.001< P <0.01, NS P >0.1. ( d ) Ubiquitination profile of E2F1 in HeLa transfected with Control (Co), PRMT-1 or PRMT-5-siRNA and pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt. E2F1 was IP using anti-HA antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene and the efficiency of siRNA were checked by western blot analysis. β -Actin was used as loading control

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Methylation, Western Blot, Transfection, Construct, Control, Ubiquitin Proteomics, Expressing, Activity Assay, Luciferase, Plasmid Preparation, Concentration Assay

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: K63 ubiquitination of E2F1 is cell cycle regulated. ( a ) Western blot analysis of E2F1, cyclin A and cIAP1 in U2OS cells transfected with cyclin A siRNA±cIAP1 siRNA. HSC70 was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA±cIAP1 siRNA, then 24 h later with pCMV-3HA-E2F1 and His-tagged ubiquitin wt. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. ( c ) Western blot analysis of the expression of 3HA-E2F1 wt or K161/164 R mutant and cyclin A in HeLa cells transfected with cyclin A siRNA. β -Actin was used as loading control. ( d ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA, then 24 h later with pCMV-3HA-E2F1 or the K161/164 R mutant and His-tagged K63-only ubiquitin. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both samples transfected with control siRNA. ( e ) Western blot analysis of cIAP1 E2F1, cyclin E, Rb, phospho (S780, S807/811) Rb in U2OS cells transfected with cIAP1-encoding construct. HSC70 was used as loading control. ( f ) Schematic representation of the regulation of E2F1. In the G1 phase of cell cycle, E2F1 is complexed to Rb. In the S phase, E2F1 is first methylated on arginine residue par PRMT, then K63-ubiquitinated on K161/164 in a cIAP1-dependent manner, leading to stabilization and activation of the protein. In the late S, cyclin A inhibits K63 ubiquitination of E2F1, binds to and phosphorylates E2F1 and promotes UPS-mediated degradation

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Expressing, Mutagenesis, Construct, Methylation, Residue, Activation Assay

Journal: bioRxiv

Article Title: ZFAND6 is a subunit of a TRAF2-cIAP E3 ubiquitin ligase complex essential for mitophagy

doi: 10.1101/2024.03.27.586763

Figure Lengend Snippet: A) Confocal microscopy with fluorescently conjugated antibodies to pUb using WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 hr. in the presence of 100 μM leupeptin. Scale bars represent 5 μm. Data presented as mean ± SEM. ( B, C) Co-IP assays were performed with lysates from 293T cells transfected with the indicated plasmids. Immunoprecipitations and western blotting were performed with the indicated antibodies to examine ZFAND6-TRAF2 (B) and ZFAND6-cIAP1 (C) interactions. (D) Co-IP assay was performed with lysates from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for the indicated times. IPs were performed with either isotype control antibody or TRAF2 antibody. Western blotting was performed with the indicated antibodies. Unpaired Student’s t-test. ****P<0.0001.

Article Snippet: ZFAND6-Flag-Myc was purchased from Origene. pEBB-HA cIAP1 was a gift from Colin Duckett (Addgene plasmid# 38232; http://n2t.net/addgene:38232 ; RRID:Addgene_38232). lentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid# 52691 ; http://n2t.net/addgene:52691 ; RRID:Addgene_52691). pUltra-Hot was a gift from Malcolm Moore (Addgene plasmid# 24130 ; http://n2t.net/addgene:24130 ; RRID:Addgene_24130). pUltra-Hot-ZFAND6 was generated by subcloning a ZFAND6 PCR product digested with Xba1 (New England Biolabs; R0145S) and BamH1-HF (New England Biolabs; R3136S) into pUltra-Hot.

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

doi: 10.1074/jbc.m110.125955

Figure Lengend Snippet: FIGURE 1. cIAP1 potently blocks cytochrome c-dependent apoptosome activation of procaspase-3-like activity but does not affect already activated caspase-3-like activity. A, schematic of wild-type cIAP1 and truncation mutants. BIR domains and CARD motifs mediate protein-protein interactions. The UBA motif enables binding of Lys-63-linked polyubiquitin chains. RING binds ubiquitin-conjugating enzymes. B, inhibition of the cytochrome c-dependent apo- ptosome by cIAP1 and the BIR2-BIR3 segment of cIAP1. Cytochrome c (Cc)-dependent apoptosome activation of caspase-3 was assayed using an S100 preparation and the indicated concentrations of GST-cIAP1 and the BIR2-BIR3 segment of cIAP1. After a 30-min incubation of S100 with cytochrome c and ATP as described under “Experimental Procedures,” the apoptosome reaction was stopped by dilution with caspase assay buffer. Caspase-3 activity was deter- mined with Ac-DEVD-AMC as substrate. C, effect of IAPs on already activated caspase-3-like activity. The caspase-3 activity of the S100 fraction was activated by the addition of cytochrome c and ATP in the absence of an added IAP. Caspase activation was stopped by diluting a sample of the apoptosome reaction by 10-fold into caspase assay buffer that contained the indicated micromolar concentration of each GST-IAP. Values are means S.E. (n 3). Control activity was 12.2 1.3 nmol of AMC produced per mg/min. *, statistically significant effect of XIAP (p 0.01) by analysis of variance (GraphPad Prism 5).

Article Snippet: Recombinant human cIAP1 (amino acids 144–356) with a C-terminal 15-amino acid AviTag was obtained from Sino Biological Inc.His10-cIAP1 andSmac56-His6were purchased from R&D Systems.

Techniques: Activation Assay, Activity Assay, Protein-Protein interactions, Binding Assay, Ubiquitin Proteomics, Inhibition, Incubation, Caspase Assay, Concentration Assay, Control, Produced

Journal: Journal of Biological Chemistry

Article Title: cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

doi: 10.1074/jbc.m110.125955

Figure Lengend Snippet: FIGURE 2. cIAP1 and XIAP prevent the processing of procaspase-3 by the cytochrome c-dependent apoptosome but have no effect on the proc- essing of procaspase-9. A, Western blot analysis of the effects of IAPs on the processing of procaspase-9 and -3. The apoptosome reaction was carried out with S64 from Apaf-1-transfected cells at 1.5 g/l for 30 min at 37 °C in the presence or absence of bovine cytochrome c (Cc; 20 g/ml) plus 1 mM ATP. A sampleoftheapoptosomereaction(24gofprotein)wasusedforSDS-PAGE (12% gel), and Western analysis of caspase-9 and -3 was done as described under “Experimental Procedures.” After immunostaining caspase-9 with the mouse monoclonal antibody, the membrane was reprobed with rabbit monoclonal antibody to cleaved caspase-3. The apoptosome reaction was carried out in the absence of cytochrome c () or in the presence of cyto- chrome c plus the indicated IAP. The concentrations of GST-Survivin, GST- Livin, GST-cIAP1, and GST-XIAP were 1.8, 1.5, 0.27, and 0.13 M, respectively. B, autoradiogram of the time course of 35S-labeled procaspase-9 processing in the presence of GST (gst only) or GST-cIAP1 (gst-cIAP1). The 20-l apo- ptosome reaction included 1.0 g/l S64 from Apaf-1-transfected cells and 1 l of 35S-labeled procaspase-9 and was incubated for the indicated intervals at 37 °C in the presence or absence of bovine cytochrome c (100 g/ml) plus 1 mM ATP. The reactions contained 500 nM purified GST or 150 nM GST-cIAP1 as indicated. After the indicated intervals, 10 l of the reaction was fraction- ated by SDS-PAGE (15% gel) and autoradiographed to visualize 35S-labeled p47 procaspase-9 and the p35 large subunit.

Article Snippet: Recombinant human cIAP1 (amino acids 144–356) with a C-terminal 15-amino acid AviTag was obtained from Sino Biological Inc.His10-cIAP1 andSmac56-His6were purchased from R&D Systems.

Techniques: Western Blot, Transfection, Immunostaining, Membrane, Labeling, Incubation, Purification, SDS Page

Journal: Journal of Biological Chemistry

Article Title: cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

doi: 10.1074/jbc.m110.125955

Figure Lengend Snippet: FIGURE 3. The procaspase-9 D315A mutant, but not the D330A mutant, reverses the inhibition of the cytochrome c-dependent apoptosome by cIAP1. A, schematic of human procaspase-9 and the positions of the autocat- alytic cleavage site (Asp-315) and the caspase-3 cleavage site (Asp-330) within the linker region (LR) between the large and small subunits of mature caspase-9. B, autoradiogram showing the processing of 35S-labeled p47 pro- caspase-9 mutants (D315A and D330A) by the apoptosome. The experiment was done as described in the Fig. 2B legend except the 35S-labeled pro- caspase-9 used was wild-type or the indicated point mutant. C, the pro- caspase-9 D315A mutant, but not the D330A mutant, prevents cIAP1 inhibi- tion of the apoptosome. The cytochrome c (Cc)-dependent apoptosome activation of procaspase-3 was assayed as described for B in the presence (Œ, f, and F) or (E) absence of 500 nM GST-cIAP1. The indicated D315A or D330A mutant of procaspase-9 (cas-9; 4 l of TNT reaction/20 l of apoptosome reaction) was added to the apoptosome reaction. Samples (4 l) of the apo- ptosome reaction were removed after the indicated intervals, diluted 10 times with caspase assay buffer, and assayed for caspase-3-like activity with Ac-DEVD-AMC as substrate. Values are means S.E. (n 3) for AMC pro- duced (nmol/mg of S64 protein).

Article Snippet: Recombinant human cIAP1 (amino acids 144–356) with a C-terminal 15-amino acid AviTag was obtained from Sino Biological Inc.His10-cIAP1 andSmac56-His6were purchased from R&D Systems.

Techniques: Mutagenesis, Inhibition, Labeling, Activation Assay, Caspase Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

doi: 10.1074/jbc.m110.125955

Figure Lengend Snippet: FIGURE 4. Smac prevents and reverses cIAP1-evoked inhibition of pro- caspase-3 activation. A, mature Smac56 (amino acids 56–239) prevents cIAP1 from inhibiting the cytochrome c-dependent apoptosome. Values are means S.E. (n three or four experiments). Cytochrome c (Cc)-dependent apoptosome activation was assayed with 1 g/l S100 fraction in the pres- ence of 40 nM GST-cIAP1 and the indicated concentration of Smac56-His6. B, mature Smac56 reverses the cIAP1 inhibition of the apoptosome. Values are means S.D. (n 3–7) for six different experiments with S64 (1.5 g/l) from Apaf-1-transfected cells. Smac56-His6 was added as indicated to 200 nM at 15 min (f) to the apoptosome reaction that received 250 nM GST-cIAP1 at 5 min (‚). Other apoptosome reactions received no GST-cIAP1 (F) or 250 nM GST-cIAP1 at 0 min () or 10 min (Œ). Samples (4 l) of the apoptosome reaction were removed after the indicated intervals, diluted 10 times with caspase assay buffer, and assayed for caspase-3-like activity with Ac-DEVD- AMC as substrate.

Article Snippet: Recombinant human cIAP1 (amino acids 144–356) with a C-terminal 15-amino acid AviTag was obtained from Sino Biological Inc.His10-cIAP1 andSmac56-His6were purchased from R&D Systems.

Techniques: Inhibition, Activation Assay, Concentration Assay, Transfection, Caspase Assay, Activity Assay