Caspase-3s Inhibits Apoptosome Assembly Figure 3. Caspase-3s influences the localization of procaspase-3. Twenty-four hours after GFP-coupled procaspase-3 (C3-GFP) or YFP- coupled caspase-3s (C3s-YFP) transfection in parental MCF-7 cells (A and B respectively) or in non tagged caspase-3s (C3s) or procaspase-3 (C3) stably transfected MCF-7 cells (C and D respectively) MCF-7 cells were treated or not with etoposide, vinblastine or methotrexate for 10 hours. Cells were scanned in phase contrast and with a GFP or YFP filter under UV light. Nuclei were delimited by dotted lines. The nulear staining quantification is presented in Table S4. doi:10.1371/journal.pone.0029058.g003 location was not influenced by any treatment (Figure 3B, second, third and fourth lines). When non tagged caspase-3s was stably expressed in MCF-7 cells before transient expression of GFP-tagged procaspase-3, the fluorescent procaspase-3 was located in both nucleus and cytoplasm and exposure to the tested drugs did not change this subcellular repartition (Figure 3C). As expected, when non tagged procaspase-3 was stably expressed in MCF-7 cells before transient expression of YFP-tagged caspase-3s, the fluorescent caspase-3s was located in both nucleus and cytoplasm and again, exposure to the tested drugs did not change significantly this subcellular repartition (Figure 3D). The fluorescent nuclear staining quantification is shown in Table S4. Caspase-3s structure predicts an absence of catalytic activity To understand why caspase-3s has no catalytic activity, we studied its protein sequence and structure. As shown in Figure 4A, caspase-3s has no catalytic site compared with procaspase-3, and especially the cysteine residue that is crucial for the catalytic activity of caspases. Moreover, caspase-3s does not possess the cleavage site between the small and the large subunit, resulting in an absence of cleavage into two subunits. Finally, in the caspase- 3s, there are no sites for binding of caspase inhibitors such as Ac- DEVD-CHO [27], XIAP [28]… However, caspase-3s possesses the same pro-domain as procaspase-3, but, contrary to procas- pase-3, caspase-3s pro-domain is not cleaved. Indeed, the activation of procaspase-3 is sequential. The first step is the cleavage between the large and the small subunits. Once this cleavage is done, the large+small subunit complex induces the cleavage of the pro-domain, involving the activation of caspase-3. Thus, if the protein is not divided in two subunits, the pro- domain can not be cleaved. Therefore, the absence of caspase-3s pro-domain cleavage may be explained by the absence of caspase-3s cleavage in two subunits. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29058