Caspase-3s Inhibits Apoptosome Assembly Figure 4. Protein sequence and 3D structure of procaspase-3 and caspase-3s. A. Alignment of procaspase-3 (C3) and caspase-3s (C3s) amino acid sequences. Prodomain is framed, amino acids forming the catalytic site are in bold blue, interaction sites with caspase-3 inhibitors are in bold green [27–28], cleavage site between small and large subunits are in bold and underlined [28], aspartate residue cleaved during activation are in bold red [29]. B. 3D structure of procaspase-3 and caspase-3s. Bold arrows represent the end of common sequence between both proteins and pink asterisks show the b-sheets and a-helixes present in procaspase-3 and absent in caspase-3s. doi:10.1371/journal.pone.0029058.g004 The difference between pro-caspase-3 and caspase-3s sequences has a strong impact on the 3D structure. In fact, when the pro- caspase-3 3D structure was compared with the caspase-3s one, it appeared that caspase-3s lacked 2 b-sheets and 2 a-helixes compared with procaspase-3 (Figure 4B). These structures are necessary for the catalytic activity of caspase-3. Caspase-3s interacts with procaspase-3 and prevents its interaction with apoptosome To understand the apoptosis inhibition mechanism of caspase-3s, co-immunoprecipitation experiments were performed in HBL100 cells stably transfected with YFP-coupled caspase-3s. Using an anti- caspase-3 monoclonal antibody that targets an epitope located in the short subunit of caspase-3, detecting only procaspase-3 and active caspase-3 but not caspase-3s, we observed, upon exposure to etoposide and methotrexate, caspase-3s interacted with procaspase- 3 (Figure 5A). Moreover, co-immunoprecipitation experiments with an anti-GFP antibody (that recognizes caspase-3s tag) demonstrated that, in cells exposed to etoposide or methotrexate, caspase-3s-YFP could pull down procaspase-3 (Figure 5B). Then, we wondered whether the interaction of caspase-3s with procaspase-3 prevents pro-caspase-3 activation by inhibiting PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29058