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。

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571336/
• 关键词: Ubiciultin; Brain ischemia; Ubipuitinated proteins; Affinity Durification; Immunoprecipitation; Proteolysis; Ubiquitin-conjugating enzymes; UBE2D2; UbE2D2; 神経細胞死; 蛋白分解; ユビキチン化蛋白質; Ubiciultin; Brain ischemia; Ubipuitinated proteins; Affinity Durification; Immunoprecipitation; Proteolysis; Ubiquitin-conjugating enzymes; UBE2D2; UbE2D2; 神経細胞死; 蛋白分解; ユビキチン化蛋白質

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

为了深入了解由缺血再灌注引起的神经元损伤和死亡的机制，我们试图纯化泛素蛋白蛋白缀合物，这些偶联物因缺血性压力而增加，这些小鼠的脑皮质会增加，这些小鼠的脑皮质受到了1小时的暂时性中性脑部脑动脉封闭，然后再生。实验得出以下结果。 （1）在再灌注0-10 h期间，用8 m尿素（尿素溶解）制备的馏分中的泛素化蛋白水平增加。 （2）优化了用于纯化尿素溶解泛素化蛋白的方案。简而言之，将尿素溶剂分数透析为4 m尿素。从所得蛋白（100-250 mg）中，通过对固定抗泛素抗体的亲和力色谱法纯化泛素化蛋白（0.1-0.4 mg），其回收率出色（几乎100％）。 （3）开发了一种鉴定纯化蛋白质中泛素化底物的方法。简而言之，蛋白质被内蛋白酶消化为…更多的P-N，在这些消化中，我们专注于与泛素（UCP）相对应的泛素的58-76片段，因为预计UCP有望在cystrate蛋白和互化蛋白链接区域携带替代蛋白质的肽片段。通过用抗UCP抗体进行免疫沉淀分离含有UCP的肽片段，并通过反相HPLC进一步分离，然后进行氨基酸测序。 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增加了泛素化的CDK5水平，并改变了CDK5在皮质中的细胞定位。 （5）从缺血性皮质的水溶性馏分中鉴定出Ube2d2（Ube2d2）的泛素 - thioester，但在对照皮层中未发现。这些结果表明，缺血性压力可能通过UBE2D2介导的途径诱导CDK5泛素化，涉及神经元损伤。较少的