Aldini Giancarlo, Carini Marina, Vistoli Giulio, Shibata Takahiro, Kusano Yuri, Gamberoni Luca, Dalle-Donne Isabella, Milzani Aldo, Uchida Koji
Istituto di Chimica Farmaceutica e Tossicologica Pietro Pratesi, Faculty of Pharmacy, University of Milan, I-20131 Milan, Italy.
Biochemistry. 2007 Mar 13;46(10):2707-18. doi: 10.1021/bi0618565. Epub 2007 Feb 13.
A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.
采用蛋白质组学方法在人神经母细胞瘤SH-SY5Y细胞中鉴定15-脱氧-Δ12,14-前列腺素J2(15d-PGJ2)的蛋白质靶点。通过使用生物素化的15d-PGJ2,发现β-肌动蛋白是主要的加合蛋白;至少还有12种蛋白质被鉴定为较小的生物素阳性斑点,属于不同的功能类别,包括糖酵解酶(烯醇化酶和乳酸脱氢酶)、氧化还原酶(胆红素还原酶)和一种真核调节蛋白(14-3-3γ)。15d-PGJ2诱导肌动蛋白丝网络发生明显的形态变化,特别是促进F-肌动蛋白解聚,蛋白质印迹分析证实了这一点。通过质谱方法,我们发现15d-PGJ2与分离的G-肌动蛋白以1:1的化学计量比反应,并通过迈克尔加成机制选择性地结合Cys374位点。计算研究表明,15d-PGJ2的共价结合诱导肌动蛋白结构发生显著的解折叠,特别是15d-PGJ2使肌动蛋白亚结构域2和4发生扭曲,这两个亚结构域定义了核苷酸结合位点,阻碍了核苷酸交换。通过聚合测量研究了15d-PGJ2对G-肌动蛋白的功能影响:在15d-PGJ2存在的情况下,形成的F-肌动蛋白量减少,芘基肌动蛋白荧光强度增加,因为随着15d-PGJ2浓度的增加,主要影响的是肌动蛋白聚合的最大程度,而对反应的初始速率影响可忽略不计。总之,这里报道的结果深入了解了15d-PGJ2作为细胞毒性化合物在神经元细胞功能障碍中的作用。肌动蛋白是15d-PGJ2的主要细胞内靶点,它通过迈克尔加成特异性地结合到Cys374,导致蛋白质构象变化,这可以解释肌动蛋白细胞骨架的破坏、F-肌动蛋白解聚和G-肌动蛋白聚合受损。