Anamorsin, a novel caspase-3 substrate in neurodegeneration [Protein Synthesis and Degradation]

June 27th, 2014 by Yun, N., Lee, Y. M., Kim, C., Shibayama, H., Tanimura, A., Hamanaka, Y., Kanakura, Y., Park, I.-S., Jo, A., Shin, J.-H., Ju, C., Kim, W.-K., Oh, Y. J.

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high-throughput screening method to identify novel substrates for caspase-3 in neuronal cell line. Critical steps in our strategy consist of 2-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell-death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25 kDa and 10 kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among 4 tetrapeptide consensus motifs DXXD existed in anamorsin, we mapped at DSVD(209)/L for a specific cleavage site for caspase-3. Intriguingly, the 25 kDa cleaved fragment of anamorsin was also detected in postmortem brains of AD and PD patients. While the RNA-interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of a full-length anamorsin into anamorsin knockout stromal cell line conferred cells resistance to staurosporine-induced caspase activation, indicating the anti-apoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates during neurodegenerative processes.