Recombinant human calpain-5 was expressed in insect cells using a baculovirus system. The expressed calpain-5 was purified by both traditional chromatography and by affinity-column chromatography. Both methods yielded active protease. Calpain-5 displayed very limited hydrophobicity. This indicated that calpain-5 is not a membrane binding protein. Calpain-5 had pI of 8.3. The recombinant calpain-5 also exhibited calcium-dependent proteolytic activity. The calculated calcium requirement for half-maximal activity was 9.6 mM when incubated at 37��C and 26.5 mM when incubated at 30��C. Compared to traditional calpains, which require less than 1 mM calcium for half-maximal activity, calpain-5 exhibited weaker proteolytic activity. This is an unusual observation because calpain-5 lacks the typical calcium-binding domain of the calpains and implied that other calcium-binding region of the protein account for calcium-binding and sensitivity. Our results also showed that calpain-5 was different from traditional calpains because its activity was higher at 37��C compared to 30��C and remained active at 37��C for more than 2 hours. This differs from traditional calpains which display better proteolytic activity at lower temperatures and become inactive within 30 minutes of incubation in 37��C. Calpain-specific inhibitors, calpastatin and E64, did not inhibit calpain-5. Only one calcium-binding inhibitor, PD150606, inhibited calpain-5 proteolytic activity. These results confirmed that calpain's calcium-binding domain is important in calpastatin binding and calpain-5 possesses other calcium-binding regions. Calpain-5 was able to degrade spectrin, a ubiquitous cytoskeletal protein. This indicates that calpain-5 might have a role in cell remodeling. Finally, calpain-5 has the ability to degrade itself. It is not clear if this is the result of inter- or intra-molecular proteolysis and whether this leads to activation of the protein or is, instead, the first step in its degradation. Calpain-5 is expressed at highest concentrations in testis, brain, liver and gastrointestinal tract. It is not clear why these tissues require a unique calpain. Calpain-5 may provide these tissues with an additional calcium-dependent proteolytic activity which is not regulated by calpastatin and which could participate in cytoskeletal protein turnover. / Graduation date: 2004

The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells. / Graduation date: 2002

Graduation date: 2006 / We hypothesized that calpain activity is elevated in response to muscle damage. To test this hypothesis, we examined the degradation of α-fodrin into its 150 and 145 kDa fragments following either 20 eccentric or isometric contractions. In addition, experiments were performed in the presence or absence of E-64-d, a calpain inhibitor. Both EDL and SOL muscles displayed significant differences (p<0.003 and p<0.002 respectively) between the raw and normalized 150 and 145 kDa α-fodrin fragments of the DMSO + E-64-d compared to the other bath treatments. Based on our model of exercise-induced muscle damage, we expected to see greater levels of 150 and 145 kDa α-fodrin fragments in those muscles that performed the eccentric protocol. However, there was no evidence that eccentric muscle damage increased the levels of 150 and 145 kDa α-fodrin fragments over the levels observed in the isometric trials. These findings suggest that the magnitude of damage was insufficient to activate calpains.