Calpain‐mediated truncation of dihydropyrimidinase‐like 3 protein (DPYSL3) in response to NMDA and H₂O₂ toxicity

Abstract

Dihydropyrimidinase-like protein 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and, possibly, neuronal regeneration. In primary cortical cultures, glutamate (NMDA) excitotoxicity and oxidative stress (H 2 O 2 ) caused the cleavage of DPYSL3, resulting in the appearance of a doublet of 62 kDa and 60 kDa. Pre-treatment of cell cultures with calpain inhibitors, but not caspase 3 inhibitor, before exposure to NMDA or H 2 O 2 completely blocked the appearance of the doublet, suggesting calpain-mediated truncation. Furthermore, in vitro digestion of DPYSL3 in cell lysate with purified calpain revealed a cleavage product identical to that observed in NMDA-and H 2 O 2 -treated cells, and its appearance was blocked by calpain inhibitors. Analysis of the DPYSL3 protein sequence revealed a possible cleavage site for calpain (ValArg-Ser) on the C-terminus of DPYSL3. Collectively, these studies demonstrate for the first time that DPYSL3 is a calpain substrate. The physiological relevance of the truncated DPYSL3 protein remains to be determined. Keywords: calpain, collapsin response-mediated protein-4, dihydropyrimidinase-like protein 3, dihydropyrimidinase related protein 3, N-methyl-D-aspartate, reactive oxygen species.

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