Date of Award
Level of Access
Master of Science (MS)
Dorothy E. Croall
Second Committee Member
Robert E. Gundersen
Third Committee Member
Julie A. Gosse
Calpains are a family of proteins that are involved in diverse biological functions, with the proteolytically-active isoforms calpain-1 and calpain-2 being the most commonly studied. Calpains contribute to multiple cellular processes such as migration, adhesion, cell cycle regulation, apoptosis and wound repair through the limited proteolysis of target substrates. Additionally, uncontrolled activation of these proteases may also contribute to pathologies such as muscular dystrophy, Alzheimer's disease, multiple sclerosis and several forms of cancer. Anti- calpain therapies may become viable treatment options for these diseases, although the biological consequences of such treatments are not well understood. Future studies are therefore required to more accurately understand calpain's contributions to pathophysiological processes by studying its interactions with other proteins.
Several methods currently exist for detecting calpain protein-protein interactions (PPIs). Proteolytic studies can identify substrates by measuring calpain-dependent cleavage, but only in the presence of high (i.e. above physiological) calcium levels. Co-Immunoprecipitation (co-IP) studies can also detect calpain PPIs, even in calcium-free conditions, but the oxidizing conditions required for this technique may alter calpain's structure and therefore affect PPI formations.
This study details the development of an alternative method for identifying calpain interactors using affinity-purification and mass spectroscopy (AP-MS) with the calpain-2 isoform. This in vitro method consists of the affinity purification of recombinant calpain-2 'bait' in order to co-capture interacting 'prey' proteins, followed by the identification of proteins using mass spectroscopy. The bait was fused with a small amino acid tag recognized by the E.coli enzyme BirA ligase for the in vitro addition of a biotin molecule; this allowed for the affinity purification of calpain-2 bait using a streptavidin-coated matrix. Due to the high affinity of the biotin - avidin interaction, this method is amenable to different binding conditions, such as the use of calcium, calcium chelators, reducing agents and detergents. As such, this technique is able to screen for interactions that would be missed using other methods such as proteolytic digestions and co-IP experiments.
The calpain-2 affinity purification method was successful in capturing interacting proteins. The purified functional inhibitory domain of calpain's endogenous inhibitor, calpastatin, was captured by the calpain-2 bait immobilized on streptavidin resin, validating the structural integrity of the bait. Additionally, preliminary AP-MS experiments using cultured bovine aortic endothelial cells as a source of mammalian proteins identified 24 calpain-2 interacting partners in the presence of calcium. Interestingly, 23 proteins were also captured by calpain-2 under calcium-free conditions, suggesting that such interactions are possible. In a separate experiment using developing zebrafish larvae as a source of proteins, 38 interactors were also captured by the calpain-2a bait, showing that this method can be adapted for different model organisms. The confirmation of true calpain-2 interactions will require repeat AP-MS trials as well as a combination of in vitro and in vivo PPI validation techniques. Future AP- MS experiments may also utilize different sources of prey proteins to investigate specific questions, such as the use of human umbilical vein endothelial cells to search for calpain-2 interactions involved in cardiovascular processes.
Hoffman, Jordan, "Developing an Affinity Purification - Mass Spectrometry Strategy to Identify Novel Calpain-2 Interacting Proteins" (2013). Electronic Theses and Dissertations. 2498.