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W. Li, N. B. Caberoy, G. Alvarado; Mapping of Substrate Degradome of Calpain by a New Technology of Functional Proteomics. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1902.
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Calpain plays a critical role in cell death in retinal diseases, including retinitis pigmentosa and glaucoma. The pathways regulated by calpain are traditionally elucidated on a case-by-case basis with daunting challenges. Consequently, calpain-regulated pathways in retinal diseases are poorly defined. The purpose of this study is to elucidate substrate degradome of calpain to facilitate the delineation of its role in retinal cell death.
Newly-developed open reading frame (ORF) phage display was used as a technology of functional proteomics to elucidate calpain substrates. ORF phage display cDNA library with C-terminal biotin tag was generated from mouse eye, bound to immobilized streptavidin, washed and eluted with calpain 2. Eluted phages were amplified and used as input for the next round of selection. After three rounds of functional selection, individual phage clones were analyzed for their activity as the substrates of calpain 2, identified by sequencing and independently verified.
New substrates of calpain 2, including Son cell proliferation proteins (Son), CCR4-NOT transcription complex subunit 3 (Cnot3), Ring finger protein 146 (Rnf146), catenin delta 2 (Ctnnd2), caldesmon 1 (Cald1) and thymopoietin (Tmpo), were systematically and efficiently elucidated by ORF phage display. Their roles as calpain substrates were independently verified with purified recombinant substrate proteins.
These data demonstrated that calpain 2 has broad substrate specificities in the eye and may regulate a number of pathways during retinal apoptosis. Moreover, ORF phage display as a powerful technology for unbiased mapping of substrate degradomes for calpain and other proteases will provide in-depth understanding of protease biology in ocular physiology and disease pathogenesis.
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