The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation

Abstract

CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3's autolytic activity and the enzyme's function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype-phenotype links in LGMD2A.calpain | skeletal muscle | muscular dystrophy | complementation | protease

Rbfox Splicing Factors Maintain Skeletal Muscle Mass by Regulating Calpain3 and Proteostasis

“…We found that two consecutive alternative exons (exons 15 and 16) in Capn 3 transcripts were almost completely skipped in Rbfox- DKO muscle and are likely direct Rbfox targets (Figure 4C). Auto- lytic cleavage within the IS1 region enables proteolytic Capn3 activity, and cleavage within the IS2 region of Capn3 is required for complete degradation and inactivation of Capn3 (Figure S4C) (Ono et al, 2014). Skipping of exons 15 and 16 in Rbfox-DKO muscle likely produces a Capn3 isoform that lacks the cleavage site within the IS2 region, thereby stabilizing active Capn3 and causing it to accumulate.…”

“…Several studies have used calpastatin as a Capn3 substrate (Ono et al, 2004, 2014). Calpastatin is also an endogenous inhibitor of Capn1 and Capn2; we predicted that reduced calpastatin levels in Rbfox DKO would lead to increased Capn1 and Capn2 activity.…”

“…It has been predicted that over 1,000 proteins are calpain targets, yet fewer than 100 substrates have mapped calpain cleavage sites because cal-pain-cleaved fragments are short-lived (Ono and Sorimachi, 2012; Piatkov et al, 2014). Despite the critical role Capn3 plays in skeletal muscle biology, it is not clear which proteins are in vivo substrates for Capn3; the majority of its substrates have been identified through biochemical or cell culture studies (Huang and Forsberg, 1998; Nakajima et al, 2006; Ono et al, 2004, 2014; Takamure et al, 2005; Taveau et al, 2003; Zhang and Bhavnani, 2006). We found that levels of two of the previously identified substrates, calpastatin and alpha-spectrin, are reduced within 7 days after the induction of Rbfox knockout.…”

Integrative variants, haplotypes and diplotypes of the CAPN3 and FRMD5 genes and several environmental exposures associate with serum lipid variables

“…CAPN3 encodes CAPN3, a heterodimer consisting of a large and a small subunit, is a major intracellular protease24, although its function has not been well established. Mutations in this gene25 are associated with lipid metabolism.…”

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…3C) and even after scaling-up the expression, the amount of full length ADGB was insufficient for further biophysical characterization. This truncation is most probably a result of proteolysis by intracellular proteases or by auto-cleavage by the N-terminal calpain-like domain, a phenomenon commonly observed among calpains [53][54][55][56]. To further evaluate if ADGB autolytic activity would be the source of this potential auto-cleavage we added three different protease inhibitors to the growth medium; the cysteine protease inhibitor E64, the acid protease inhibitor Pepstatin A and the calpain specific Calpain II inhibitor.…”