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

泛素-蛋白缀合物积累对脑缺血引起的细胞损伤和死亡的意义的研究。

基本信息

批准号：
14571336
负责人：
TAKADA Koji
金额：
$ 2.43万
依托单位：
JIKEI UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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

为了深入了解缺血-再灌注诱导的神经元损伤和死亡的机制，我们试图纯化泛素-蛋白质缀合物，这是增加了缺血应激，从小鼠大脑皮层，这是经过短暂的大脑中动脉闭塞1小时，然后再灌注。实验产生了以下结果。(1)再灌注0-10小时，用8 M尿素（尿素可溶性）制备的组分中的泛素化蛋白水平增加。(2)纯化尿素可溶性泛素化蛋白的方案进行了优化。简言之，将尿素可溶性级分针对4 M尿素透析。从所得蛋白质（100-250 mg）中，通过在固定化的抗泛素抗体上的亲和层析纯化泛素化蛋白质（0.1-0.4 mg），具有优异的回收率（几乎100%）。(3)建立了一种鉴定纯化蛋白中泛素化底物的方法。简言之，用内切蛋白酶As消化蛋白质 ...更多信息 p-N，并且在这些实验中，我们集中于对应于泛素（UCP）的C-末端部分的泛素片段58-76，因为UCP预期携带底物蛋白的肽片段和链的泛素间连接区域。用抗UCP抗体免疫沉淀法分离含有UCP的肽段，并通过反相HPLC进一步分离，随后进行氨基酸测序。最后，我们从尿素可溶性组分中鉴定了泛素化的细胞周期蛋白依赖性激酶5（CDK 5）和K48连接的泛素链，这两种蛋白在缺血皮质中更丰富（再灌注5小时）比正常皮质：（4）通过免疫组织化学和抗CDK 5抗体的免疫印迹分析，我们发现缺血应激增加了泛素化CDK 5的水平，并改变了CDK 5在皮质中的细胞定位。(5)泛肽硫酯与UBE 2D 2，泛肽共轭酶的成员，被确定从缺血皮质的水溶性馏分，但没有发现在控制皮质。提示缺血应激可能通过UBE 2D 2介导的途径诱导CDK 5泛素化，进而导致神经元损伤。少