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

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.

Recombinant anthrax lethal toxin inhibits cell motility and invasion in breast cancer cells through the dysregulation of Rho GTPases.

Breast cancer is the leading cause of cancer-associated death among women worldwide. Targeting breast cancer cell metastasis is an important therapeutic approach. The MAPK pathway is a key cell signaling pathway that plays a pivotal role in cellular invasion and migration. Numerous studies have identified the MAPK pathway as a way to target cell survival and motility. The present study treated MBA-MD-231 breast cancer cells with anthrax lethal toxin (LeTx), a potent MAPK inhibitor that selectively cleaves and inactivates all MEKs, as a potential therapeutic method to inhibit breast cancer cell migration. LeTx has been demonstrated to affect breast cancer cell migration. Cells treated with LeTx showed a significant decrease in motility, as observed using wound healing and random 2D motility assays. Additionally, cells treated with LeTx showed an increase in adhesion, which would explain the decrease in migration. Pull-down assays examining the activation status of the members of the Rho family of GTPases revealed an increase in RhoA activation accompanied by a decrease in Cdc42 activation following LeTx treatment. Finally, LeTx mediated a decrease in invasion using a Boyden chamber assay, which could be a result of the decrease in Cdc42 activation. The present study reported the effect of LeTx treatment on the migration, adhesion and invasion of breast cancer cells, demonstrating that this effect was associated with the dysregulation of the Rho GTPases, RhoA and Cdc42.