Dosemeci et al. (2012). Biochemical and Biophysical Research Communications. doi: 10.1016/j.bbrc.2012.10.131
…n from rat cerebral cortices was carried out as described previously [18]. Brains from adult Sprague Dawley rats were collected and frozen in liquid nitrogen within 2 min of decapitation by Pel-Freeze Biologicals (Rogers, AR). Frozen brains were thawed for 1 min in 0.32 M sucrose at 37 C and were imme- diately dissected and homogenized. For rapidly processed PSD preparations, cerebral cortices
Abstract
Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.