Studies on significance of accumulation of ubiquitin-protein conjugates with respect to cell damage and death induced by brain ischemia.

为了深入了解缺血再灌注引起的神经元损伤和死亡的机制，我们试图从小鼠大脑皮层中纯化泛素蛋白偶联物，这些泛素蛋白偶联物在缺血再灌注后1小时短暂性大脑中动脉闭塞后增加。实验产生了以下结果。(1)在再灌注0 ~ 10 h时，8 M尿素（脲溶性）制备的组分中泛素化蛋白水平升高。(2)优化了尿素可溶性泛素化蛋白的纯化方案。简单地说，尿素可溶部分在4 M尿素中透析。从所得蛋白（100-250 mg）中，通过固定抗泛素抗体亲和层析纯化泛素化蛋白（0.1-0.4 mg），回收率高（几乎100%）。(3)建立了一种鉴定纯化蛋白中泛素化底物的方法。简而言之，这些蛋白被内源性蛋白酶As…More p-N消化，在这些消化中，我们重点研究了与泛素c端部分（UCP）对应的泛素片段58-76，因为UCP可能携带底物蛋白的肽片段和泛素链的相互链接区域。采用抗UCP抗体免疫沉淀法分离含UCP的肽段，反相高效液相色谱法分离后进行氨基酸测序。最后，我们从尿素溶组分中鉴定出泛素化周期蛋白依赖性激酶5 （CDK5）和k48连接的泛素链，两者在缺血皮质（再灌注5小时）中比在正常皮质中更丰富。(4)使用抗CDK5抗体的免疫组织化学和免疫印迹分析，我们发现缺血应激增加了泛素化CDK5水平，并改变了CDK5在皮质中的细胞定位。(5)泛素硫酯与泛素偶联酶UBE2D2在缺血皮质的水溶性组分中被鉴定出来，而在对照皮质中未被发现。这些结果表明，缺血应激可能通过ube2d2介导的途径诱导CDK5泛素化，涉及神经元损伤。少

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 … More 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