Identification of novel protein targets for modification by 15-deoxy-Δ12,14-prostaglandin J2 in mesangial cells reveals multiple interactions with the cytoskeleton
Identification of novel protein targets for modification by 15-deoxy-Δ12,14-prostaglandin J2 in mesangial cells reveals multiple interactions with the cytoskeleton
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DOI：
10.1681/asn.2005030329
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发表时间：
2006-01-01
影响因子：
13.6
通讯作者：
Perez-Sala, Dolores
中科院分区：
文献类型：
--
作者：
Stamatakis, Konstantinos;Sanchez-Gomez, Francisco J.;Perez-Sala, Dolores
环戊烯酮前列腺素 15-脱氧-Delta(12,14)-PGJ(2) (15d-PGJ(2)) 已被证明对肾损伤或炎症具有保护作用。在培养的系膜细胞 (MC) 中，15d-PGJ(2) 抑制促炎基因的表达并调节细胞增殖。因此，环戊烯酮前列腺素（cyPG）被认为有望治疗肾脏疾病。 15d-PGJ(2) 的作用可能取决于或独立于其作为过氧化物酶体增殖物激活受体激动剂的作用。最近的研究表明，15d-PGJ(2) 的过氧化物酶体增殖物激活受体独立作用的一个重要决定因素是共价修饰蛋白质并改变其功能的能力。然而，迄今为止，已确定的蛋白质靶标数量有限。本文显示，15d-PGJ(2)的生物素化衍生物概括了15d-PGJ(2)对应激反应和诱导型一氧化氮合酶水平抑制的作用，并与完整MC中的蛋白质形成稳定的加合物。然后使用生物素化的 15d-PGJ(2) 来鉴定可能参与 cyPG 生物效应的蛋白质。通过二维电泳分离生物素化 15d-PGJ(2) 处理的 MC 的提取物，并通过质谱分析感兴趣的斑点。已确定的靶标包括受氧化应激调节的蛋白质，例如热休克蛋白 90 和核苷二磷酸激酶，以及参与细胞骨架组织的蛋白质，例如肌动蛋白、微管蛋白、波形蛋白和原肌球蛋白。通过抗生物素蛋白下拉证实了生物素化 15d-PGJ(2) 与多个靶标的结合。与这些发现一致，15d-PGJ(2) 诱导 MC 中波形蛋白和微管蛋白的早期重组。环戊烯酮部分和半胱氨酸的存在对于波形蛋白重排很重要。这些研究可能有助于了解 cyPG 的作用机制和治疗潜力。
The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ(2) inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ(2) may be dependent on or independent from its role as a peroxisome proliferator-activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator-activated receptor-independent effects of 15d-PGJ(2) is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ(2) recapitulates the effects of 15d-PGJ(2) on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ(2) was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ(2)-treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ(2) binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ(2) induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.