Farout Luc, Mary Jean, Vinh Joëlle, Szweda Luke I, Friguet Bertrand
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, EA 3106, IFR 117, Université Paris 7 - Denis Diderot, 2 place Jussieu, 75251 Paris cedex 05, France.
Arch Biochem Biophys. 2006 Sep 1;453(1):135-42. doi: 10.1016/j.abb.2006.02.003. Epub 2006 Feb 24.
The proteasome represents a major intracellular proteolytic system responsible for the degradation of oxidized and ubiquitinated proteins in both the nucleus and cytoplasm. We have previously reported that proteasome undergoes modification by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and exhibits declines in peptidase activities during cardiac ischemia/reperfusion. This study was undertaken to characterize the effects of HNE on the structure and function of the 20S proteasome. To assess potential tissue-specific differences in the response to HNE, we utilized purified 20S proteasome from heart and liver, tissues that express different proteasome subtypes. Following incubation of heart and liver 20S proteasome with HNE, changes in the 2D gel electrophoresis patterns and peptidase activities of the proteasome were evaluated. Proteasome subunits were identified by mass spectrometry prior to and following treatment with HNE. Our results demonstrate that specific subunits of the 20S proteasome are targeted for modification by HNE and that modified proteasome exhibits selective alterations in peptidase activities. The results provide evidence for a likely mechanism of proteasome inactivation in response to oxidative stress particularly during cardiac ischemia/reperfusion.
蛋白酶体是一种主要的细胞内蛋白水解系统，负责细胞核和细胞质中氧化和泛素化蛋白质的降解。我们之前报道过，蛋白酶体在心脏缺血/再灌注期间会受到脂质过氧化产物4-羟基-2-壬烯醛（HNE）的修饰，并且肽酶活性会下降。本研究旨在表征HNE对20S蛋白酶体结构和功能的影响。为了评估对HNE反应中潜在的组织特异性差异，我们使用了从心脏和肝脏纯化的20S蛋白酶体，这两种组织表达不同的蛋白酶体亚型。将心脏和肝脏20S蛋白酶体与HNE孵育后，评估蛋白酶体二维凝胶电泳图谱和肽酶活性的变化。在HNE处理之前和之后，通过质谱鉴定蛋白酶体亚基。我们的结果表明，20S蛋白酶体的特定亚基是HNE修饰的靶点，并且修饰后的蛋白酶体在肽酶活性方面表现出选择性改变。这些结果为蛋白酶体在氧化应激反应中失活的可能机制提供了证据，特别是在心脏缺血/再灌注期间。
