Abstract
As opposed to proteasome-mediated proteolysis that leads to protein degradation, calpain proteases carry out limited proteolytic cleavages of their substrates. The cleavage of some substrates can produce active fragments that perform functions that are different from those performed by the full-length proteins. Therefore, cleavage by calpains can operate as a posttranslational modification and increase the functional diversity of target proteins. Nevertheless, activation of protein function by calpain cleavage is still an understudied area in molecular biology. Identifying and functionally characterizing by products generated by calpain cleavage could lead to the discovery of biomarkers and the identification of novel drug targets for the treatment of human diseases. This chapter contains a workflow designed to experimentally characterize novel calpain substrates, including identification of potential calpain targets via Western blotting, characterization of calpain cleavage sites, and the study of cellular functions played by such cleaved products. We will employ MYC as an example for these experiments.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83(3):731–801. https://doi.org/10.1152/physrev.00029.2002
Ono Y, Sorimachi H (2012) Calpains: an elaborate proteolytic system. Biochim Biophys Acta 1824(1):224–236. https://doi.org/10.1016/j.bbapap.2011.08.005
Xu W, Wong TP, Chery N, Gaertner T, Wang YT, Baudry M (2007) Calpain-mediated mGluR1alpha truncation: a key step in excitotoxicity. Neuron 53(3):399–412. https://doi.org/10.1016/j.neuron.2006.12.020
Abe K, Takeichi M (2007) NMDA-receptor activation induces calpain-mediated beta-catenin cleavages for triggering gene expression. Neuron 53(3):387–397. https://doi.org/10.1016/j.neuron.2007.01.016
Vennstrom B, Sheiness D, Zabielski J, Bishop JM (1982) Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. J Virol 42(3):773–779
Conacci-Sorrell M, Ngouenet C, Eisenman RN (2010) Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell 142(3):480–493. https://doi.org/10.1016/j.cell.2010.06.037
Eisenman RN (2000) The max network: coordinated transcriptional regulation of cell growth and proliferation. Harvey Lect 96:1–32
Conacci-Sorrell M, Ngouenet C, Anderson S, Brabletz T, Eisenman RN (2014) Stress-induced cleavage of Myc promotes cancer cell survival. Genes Dev 28(7):689–707. https://doi.org/10.1101/gad.231894.113
Anderson S, Poudel KR, Roh-Johnson M, Brabletz T, Yu M, Borenstein-Auerbach N, Grady WN, Bai J, Moens CB, Eisenman RN, Conacci-Sorrell M (2016) MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation. Proc Natl Acad Sci U S A 113(37):E5481–E5490. https://doi.org/10.1073/pnas.1610994113
Branca D (2004) Calpain-related diseases. Biochem Biophys Res Commun 322(4):1098–1104. https://doi.org/10.1016/j.bbrc.2004.07.126
Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI (2000) Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26(2):163–175. https://doi.org/10.1038/79876
Ono Y, Saido TC, Sorimachi H (2016) Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov 15(12):854–876. https://doi.org/10.1038/nrd.2016.212
Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, Friedrich P (2004) On the sequential determinants of calpain cleavage. J Biol Chem 279(20):20775–20785. https://doi.org/10.1074/jbc.M313873200
Lynch DR, Gleichman AJ (2007) Picking up the pieces: the roles of functional remnants of calpain-mediated proteolysis. Neuron 53(3):317–319. https://doi.org/10.1016/j.neuron.2007.01.014
Collins GA, Goldberg AL (2017) The logic of the 26S proteasome. Cell 169(5):792–806. https://doi.org/10.1016/j.cell.2017.04.023
Lee DH, Goldberg AL (1998) Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 8(10):397–403
Mumford RA, Pickett CB, Zimmerman M, Strauss AW (1981) Protease activities present in wheat germ and rabbit reticulocyte lysates. Biochem Biophys Res Commun 103(2):565–572
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
del Carmen Lafita-Navarro, M., Conacci-Sorrell, M. (2019). Identification of Calpain-Activated Protein Functions. In: Messer, J. (eds) Calpain. Methods in Molecular Biology, vol 1915. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8988-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8988-1_12
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-8987-4
Online ISBN: 978-1-4939-8988-1
eBook Packages: Springer Protocols