| Research Abstract |
To gain insight into mechanisms of neuronal damage and death induced by ischemia-reperfusion, we attempted to purified ubiquitin-protein conjugates, which were increased by the ischemic stress, from cerebral cortices of mice, which were subjected to 1 hour transient middle cerebral artery occlusion followed by reperfusion. The experiments yielded the following results. (1) Ubiquitinated protein levels in fractions prepared with 8 M urea (urea-soluble) were increased during 0-10 h ofreperfusion. (2) Protocols for purifying urea-soluble ubiquitinated proteins were optimized. Briefly, the urea-soluble fraction was dialyzed against 4 M urea. From the resultant proteins (100-250 mg), ubiquitinated proteins (0.1-0.4 mg) were purified by affinity' chromatography on immobilized anti-ubiquitin antibody with excellent recovery rates (almost 100%). (3) A method for identifying ubiquitinated substrates in the purified proteins was developed. Briefly, the proteins were digested by endoproteinase As
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p-N, and in these digests, we focused on the fragment 58-76 of ubiquitin corresponding to C-terminal part of ubiquitin (UCP), since UCP is expected to carry peptide fragments of substrate proteins and interubiquitin linkage regions of the chains. The peptide fragments containing UCP were isolated by immunoprecipitation with anti-UCP antibody, and further separated by reverse-phase HPLC followed with amino acid sequencing. Finally, we identified ubiquitinated cyclin-dependent kinase 5 (CDK5) and K48-linked ubiquitin chain from the urea-soluble fractions, both of which were more abundant in ischemic cortex (5 h of reperfusion) than in normal cortex : (4) Using immunohistochemical and immunoblot analyses with anti-CDK5 antibody, we found that ischemic stress increased ubiquitinated CDK5 levels, and changed cellular localization of CDK5 in the cortex. (5) Ubiquitin-thioester with UBE2D2, a member of ubiquitin-conjugating enzymes, was identified from water-soluble fractions of the ischemic cortex, but not found in the control cortex. These results suggest that the ischemic stress may induce ubiquitination of CDK5 via UBE2D2-mediated pathway, involving neuronal damage. Less
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