Calpain-mediated truncation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H2O2 toxicity.
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 (H2O2) 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 H2O2 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 H2O2-treated cells, and its appearance was blocked by calpain inhibitors. Analysis of the DPYSL3 protein sequence revealed a possible cleavage site for calpain (Val-Arg-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.
Kowara
Renata
R
National Research Council, Institute for Biological Sciences, Ottawa, Ontario, Canada. Renata.Kowara@nrc-cnrc.gc.ca
Chen
Qianfa
Q
Milliken
Melissa
M
Chakravarthy
Balu
B
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000494:pharmacology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000633:toxicity*; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000633:toxicity*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
D061945:Caspase Inhibitors; C449760:Dpysl3 protein, rat; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D009419:Nerve Tissue Proteins; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D006861:Hydrogen Peroxide; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D002118:Calcium