ABSTRACT
Calpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: K.O., H.S., Y.O.; Methodology: K.O., H.S., Y.O.; Validation: K.O.; Formal analysis: K.O.; Investigation: K.O., S.H., F.S.-O., M.O., S.M.; Resources: K.O., S.H., F.S.-O., H.S., Y.O.; Data curation: K.O., S.H., F.S.-O., M.O., S.M., Y.O.; Writing - original draft: K.O., Y.O.; Writing - review & editing: K.O., Y.O.; Project administration: K.O.; Funding acquisition: K.O.
Funding
This work was supported in part by the Japan Society for the Promotion of Science KAKENHI [no.23500477 and 19H04014] and Naito Foundation to K.O.
- Received October 24, 2019.
- Accepted July 28, 2020.
- © 2020. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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