Research ReportRole of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase
Section snippets
Cleavage of GAD67 under neuronal stimulation condition
When the rat brain synaptosomes were stimulated by 55 mM K+ and 2.2 mM Ca2+ for 5 min, the extent of the conversion of fGAD67 to the truncated form was increased in comparison with the non-stimulation condition (Fig. 1A). The amounts of the truncated GAD67 were significantly increased under stimulation for an additional 5 min (Fig. 1A). When 2 mM EDTA/EGTA was present during the period of stimulation, no significant cleavage was observed 6 (Fig. 1A), suggesting that the cleavage of GAD67 in rat
Discussion
An important mechanism involved in the regulation of enzyme activity is through proteolytic cleavage of enzyme proteins. For instance, protein tyrosine phosphatases (PTPs) is regulated by a proteolytic mechanism and the cleaved proteins show higher activity (Gu and Majerus, 1996). In striatum neurons, striatal tyrosine phosphatase (STEP) is proteolytically cleaved by glutamate-induced calcium influx and calpain activation. The cleaved STEP can reach its targets and perform its function. In this
Materials
Protein inhibitor cocktail, ionomycin, μ-calpain, calpain inhibitor I, polyclonal anti-μ-calpain, polyclonal anti-m-calpain, goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP), ABTS [2,2′-azino-bis(3-ethylbenziazoline-6-sulfonic acid)], and Igepal CA 630 were purchased from Sigma (St. Louise, MO). Calpastatin peptide was purchased from Calbiochem (San Diego, CA). Lipofectamine, Opti-MEM medium, TRIZOL and the cDNA synthesis kit were from Invitrogen (Carlsbad, CA). pfu polymerase
Acknowledgments
This work was supported in part by National Institutes of Health Grant NS37851 (to J.-Y.W.), National Science Foundation grant IBN-9723079 (to J.-Y.W), the Schmidt Family Foundation and the Center of Excellence for Biomedical and Marine Biotechnology at Florida Atlantic University.
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2016, Progress in NeurobiologyCitation Excerpt :Together, these results suggest that calpain-mediated cleavage of GAD65 may downregulate GABA-mediated phasic inhibition. Studies similar to those described above for GAD65 also showed the calpain-mediated cleavage of GAD67 (Sha et al., 2008), which may decrease the synthesis of GABA, as observed in in vitro experiments using purified human GAD67 and recombinant μ-calpain. Silencing of the m-calpain gene had no effect on the level of truncated GAD in cultured neurons isolated from the whole brain, whereas knock-down of μ-calpain greatly decreased the cleavage of both GAD isoforms.
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