Fig 1: CYLD cleavage during mitochondrial apoptosis requires CASP3.(A) Schematic representation of CYLD and SPATA2. Known cleavage sites by CASP8 and MALT1 are indicated. CG, Cap-Gly; USP, Ubiquitin-specific protease. (B) Immunoblot analysis of Ctrlsg, CASP3sg, CASP8sg, or BAX/BAKsg MDA-MB-231 cells treated with ABT-737 and S63845 (A/S, 10 μM each) for 2 h. Closed and open triangles indicate full-length and cleaved proteins. (C) Immunoblot analysis of Ctrlsg and CASP3sg THP-1 cells exposed to escalating doses of A/S for 1 h. (D) Cell-free extracts prepared from THP-1 or MCF7 cells were incubated for 3 h with cytochrome c (cytc.c, 50 μg/mL) and dATP (1 mM). When indicated, extracts were preincubated for 15 min with Q-VD-OPh (10 μM). (E) THP-1 cells were treated with A/S (1 μM each) as indicated. Cell lysates were subjected to immunoprecipitation (IP) with antibodies against SPATA2 or with nonrelevant antibodies, and samples were then analyzed by immunoblotting. (F) Dose–response curves of Ctrlsg and SPATA2sg THP-1 cells treated with escalating doses of A/S (15.625, 31.25, 62.5, 125, 250, 500, and 1000 nM) for 1 h. Metabolic cell survival was assessed using CellTiter Glo (n = 4; mean ± SEM; extra sum-of-squares F test; P values for each comparison: Ctrlsg vs SPATA2sg#1: P = 0.2074, Ctrlsg vs SPATA2sg#2: P = 0.0015). (G) Immunoblot analysis of Ctrlsg and SPATA2sg THP-1 cells treated with escalating doses of A/S for 1 h. The presented immunoblots are representative of at least three independent experiments. Source data are available online for this figure.
Fig 2: CYLD is cleaved by CASP3 at aspartic acid 364.(A) In vitro cleavage of recombinant human CYLD incubated with the indicated recombinant CASPs. Closed and open triangles indicate full-length and cleaved CYLD. (B) Purified CYLD was incubated together with the indicated CASPs, and CYLD DUB activity was assessed using Lys63-Ub4 and Met1-Ub4 as substrates. Samples were incubated for 1 h, resolved by SDS-PAGE, and stained using silver stain. (C) Schematic of CYLD protein indicating the predicted CASP3 cleavage sites (D345 and D364) in addition to the known CASP8 cleavage site. The predicted and measured COOH-terminal products are also shown. (D) Alignment of CYLD amino acid sequences from multiple species. The conserved aspartic acids are highlighted in bold. (E, F) HEK293T cells were transfected with the indicated FLAG/HA-CYLD plasmids. After 24 h, cell lysates were prepared and immunoprecipitated with FLAG antibodies. Samples were eluted with FLAG peptides before incubation with recombinant CASPs as indicated. Samples were analyzed by immunoblotting as indicated. (G) Immunoblots for indicated proteins in control cells (CYLDWT) or knock-in THP-1 cells engineered to express homozygous D364A mutation (CYLDD364A), treated with ABT-737 and S63845 (A/S) for 1 h. The presented immunoblots are representative of at least three independent experiments. Source data are available online for this figure.
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