Calpain cleavage of Junctophilin-2 generates a spectrum of calcium-dependent cleavage products and DNA-rich NT1-fragment domains in cardiomyocytes

Abstract

Calpains are calcium-activated neutral proteases involved in the regulation of key signaling pathways. Junctophilin-2 (JP2) is a Calpain-specific proteolytic target and essential structural protein inside Ca2+ release units required for excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in heart failure has been reported, the precise molecular identity of the Calpain cleavage sites and the (patho-)physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently at three additional secondary Calpain cleavage sites. Moreover, we identified the Calpain-specific primary cleavage products for the first time in human iPSC-derived cardiomyocytes. Knockout of RyR2 in hiPSC-cardiomyocytes destabilized JP2 resulting in an increase of the Calpain-specific cleavage fragments. The primary N-terminal cleavage product NT1 accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+-dependent manner, closely associated with euchromatic chromosomal regions, where NT1 is proposed to function as a cardio-protective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.

New Insights into the Proteolytic Regulation of the Structural Protein Junctophilin-2 by Calpain

“…These JP2-RyR interactions may protect JP2 against Calpain cleavage by covering the Calpain cleavage sites (Figure 1). We showed that knockout of RyR2 (cardiac isoform) in 2-month-old human iPSC-derived cardiomyocytes destabilizes full-length JP2 resulting in an increase of the Calpain-specific primary cleavage fragments, while RyR2 appeared to be more resistant to Calpain cleavage than JP2 in cardiomyocyte lysates treated with Calpain-1 or -2 [10]. Hence, RyR2 seems to shield JP2 against Calpain-dependent cleavage presumably through protein-protein interactions.…”

“…The joining region, which is a long flexible linker between MORN1-6 and MORN7-8, and the divergent region connecting αH and TM are unresolved in the JP2 crystal structure [11] and AlphaFold2 predictions (UniProt protein ID: Q9BR39) [19,20], presumably because of higher intrinsic disorder [10,11]. In our recent publication, we showed that the joining region and divergent region of JP2 represent hot spots for Calpain cleavage sites using in vitro calpain cleavage assays of purified proteins [10]. Calpain preferentially cleaves mouse JP2 in the divergent region at residue R565.…”

“…Indeed, the primary and secondary Calpain cleavage sites of mouse JP2 are in close proximity to predicted PEST sequence signatures, two in the joining region and three in the divergent region. Interestingly, the PEST motif with the highest score (a.a. 565-589, score: 26.85) overlaps with the primary Calpain cleavage site of JP2 at R565 [10]. PEST sequences are frequently characterized by higher protein backbone flexibility [24], which may enable a better steric accessibility and broad conformation spectrum for diverse interactions of the joining region and divergent region.…”

“…In the joining region of JP2, several genetic variants associated with hypertrophic or dilated cardiomyopathy are clustered near the secondary Calpain cleavage site of JP2 at S164 (e.g., V153A, R156C, P158L, L159Q, T161A, S165F, S165N172delinsH, S168R, E169K, G173R) and at ~250 (e.g., R231Q, E234K, T237A, S241R, A261V, S265R) [10,11,13]. The Calpain cleavage site of mouse JP2 at S164 is conserved in human at S164 (161-TSLS↓SLRS-168) [10]. In the S165F variant, the C-terminal side of the scissile bond (P1') is mutated from serine to phenylalanine substituting a favored for a disfavored residue in P1' [23,36].…”

“…One emerging target of Calpain cleavage under pathophysiological conditions is Junctophilin-2 (JP2) [8,9]. Recently, we systematically analyzed the JP2 cleavage products as a function of Calpain-1 versus Calpain-2 proteolytic activities [10]. Together with newly…”