The identified proteins were categorized according to singular enrichment analysis of biological process

The identified proteins were categorized according to singular enrichment analysis of biological process. the regulation of the level of cytoskeleton proteins during apoptosis. Keywords:apoptosis, caspase-2, cytoskeleton protein, protein degradation, proteomics The execution of apoptotic cell death is mediated by the caspase family of cysteine proteases. After being synthesized, caspases remain as inactive zymogens in the cell, where their activities are strictly regulated by proteinprotein interactions and by proteolysis. Upon activation, caspases cleave each other’s zymogens or other Rabbit Polyclonal to MRIP cellular substrates, thus eliciting orchestrated cellular destruction.1,2Among mammalian caspase family proteins, caspase-2 is the most conserved and evolutionarily related to CED-3, theCaenorhabditis elegansprotease.3,4Yet, its biological function remains a matter of controversy. Over the past years, intense investigation of the function and activation mechanisms of caspase-2 has implicated this enzyme in stress-induced cell death pathways, including those triggered by DNA damage, microtubule destabilization and metabolic imbalance.5,6,7,8In addition, evidence for a potential role of caspase-2 in non-apoptotic pathways, including cell cycle regulation and DNA repair, is emerging (reviewed in Vakifahmetoglu-Norberg and Zhivotovsky9and Kumar10). Caspase-2 possesses features of both initiator and executioner caspases. It shares sequence homology with the initiator caspases, especially caspase-9, but compared with other known caspases, which require tetrapeptide specificity for cleavage, the preferred substrate for caspase-2 is the pentapeptide VDVAD and the predicted substrate specificity of caspase-2 is more like that of caspase-3 or -7.11,12,13,14Among all cellular proteins that are known to be cleaved by caspases, only a few have been reported to serve as substrates for caspase-2. In addition to itself, active caspase-2 has been shown to cleave CUX-1, huntingtin (Htt),ll-spectrin, Bid, Rho kinases-2 (Rock-2), PKC, plakin, HDAC4, MDM-2, ICAD, PARP and DNp63a.15,16,17,18,19,20,21,22,23,24,25,26Most of these proteins are substrates for multiple proteases, including calpains and other caspases, and the functional consequences of their cleavage by caspase-2 remain unclear. The only caspase-2-specific substrate is the peripheral Golgi membrane protein golgin-160,15which possesses a caspase-2-specific cleavage site. In addition, desmoplakin, a cytoskeletal protein, has been reported to be subject to caspase-2 cleavage.25Although the cleavage site and functional significance of this proteolysis are presently unknown, desmoplakin appears to be cleaved explicitly by caspase-2 and not by other caspases. It seems likely that caspases might exert diverse functions Dihydroxyacetone phosphate based on their substrate selectivity during various cell fates. Whether caspase-2-mediated substrate cleavage is mandatory for apoptosis and which proteins it can cleave under different circumstances are yet to be determined. Therefore, the identification of novel caspase-2 substrates remains an important goal. In this study, we investigated the effect of active recombinant human caspase-2 on different subcellular compartments using a two-dimensional gel electrophoresis (2D-DIGE) proteomics approach. We used fractionated lysates of HCT116 human colon cancer cell line and compared the appearance or disappearance of protein peak intensities following treatment with recombinant caspase-2 alone or in combination with ankyrin (DARPin AR_F8), a specific caspase-2 inhibitor.27We found that the levels of four cytoskeleton proteins, tropomyosin, profilin, stathmin-1 and myotrophin, were decreased specifically in samples treated with recombinant caspase-2. The Dihydroxyacetone phosphate degradation profiles of these cytoskeleton proteins were further confirmed by western blotting inin vitroassays and during apoptotic signaling that requires caspase-2 activation. In samples where caspase-2 activity was pharmacologically inhibited, or downregulated using siRNA, the degradation of these proteins was blunted. Nonetheless, the degradation of the analyzed cytoskeleton proteins did not depend on the direct cleavage by caspase-2. Instead, caspase-2 probed these proteins for proteasomal degradation. Although the exact mechanism by which caspase-2 focuses on these proteins to the proteasome is definitely unclear, our study suggests a novel part for caspase-2 in mediating proteasomal degradation of cytoskeleton proteins during apoptosis. == Results == == Subcellular fractionation and optimization of conditions for recombinant caspase-2 activity == To investigate whether any cytoskeleton proteins might serve as a caspase-2 substrate, we 1st fractionated HCT116 cells into cytosolic, membrane and nuclear compartments using the Qproteome Cell Compartment Kit (Number 1a). Each of these fractions was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were visualized by Coomassie staining (Number Dihydroxyacetone phosphate 1a). The purity of each fraction was verified by western blotting using antibodies against Dihydroxyacetone phosphate proteins known to.