Identifying Novel Apoptotic and Non-Apoptotic Substrates of Caspases

  • Author / Creator
    Araya, Luam Ellen
  • Caspases are a family of enzymes that regulate biological processes, such as inflammation and programmed cell death, through proteolysis. For example, in the intrinsic pathway of apoptosis, cell death signaling involves cytochrome c release from the mitochondria, which leads to the activation of caspase-9, and eventually the executioners, caspase-3 and -7. One key step in our understanding of these proteases is to identify their respective protein substrates. Whereas hundreds of substrates have been linked to caspase-3, only a small handful of substrates have been reported for caspase-9. Employing deep profiling by subtiligase N-terminomics, we present here an unbiased analysis of caspase-3 and caspase-9 substrates in native cell lysates. We identified 906 protein substrates associated with caspase-3, and 124 protein substrates for caspase-9. This is the most comprehensive list of caspase substrates reported for each of these proteases revealing a pool of new substrates that could not have been discovered using conventional interactomics approaches. Over half of the caspase-9 substrates were also cleaved by caspase-3, but often at unique sites, suggesting an evolved functional redundancy for these two proteases. Correspondingly, nearly half of the caspase-9 cleavage sites were not recognized by caspase-3. Our results suggest that in addition to its important role in activating the executioners, the role of caspase-9 is likely broader and more complex than previously appreciated, which includes proteolysis of key apoptotic substrates other than just caspase-3 and -7, and involvement in non-apoptotic pathways. Our results are well poised to aid the discovery of new biological functions for these two caspases. As well, we assessed the global proteome changes incurred in C2C12 cells following serum withdrawal-induced differentiation from myoblasts to myotubes; this differentiation process has been shown to involve the non-lethal activation of caspase-3. Our investigation indicates enrichment of key differentiation markers in harvested myotubes, and differential expression of proteins involved in rRNA processing in undifferentiated cells, and muscle contraction processes in differentiated myotubes, as expected. This experiment provides a starting block to further N-terminomics and phosphoproteomics work.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.