Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; Child Neuropsychiatry Unit, University Hospital, Azienda Ospedaliera Universitaria Senese (AOUS), Siena, Italy.
Department of Biomedical and Specialist Surgical Sciences, University of Ferrara, Ferrara, Italy.
Int J Biochem Cell Biol. 2016 Dec;81(Pt B):236-245. doi: 10.1016/j.biocel.2016.08.001. Epub 2016 Aug 2.
Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)-a major secondary product of lipid peroxidation- and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients.
雷特综合征（RTT）是一种普遍的发育障碍，主要影响女孩，发病率为每 10000 名新生儿中就有 1 例。在超过 90%的经典 RTT 病例中，一个明确的病因因素是编码甲基-CpG 结合蛋白 2（MECP2）的基因突变。我们小组最近的工作表明，这些患者存在系统性氧化应激（OxS），与临床特征的严重程度相关。我们使用来自 RTT 患者和健康受试者的新鲜分离的皮肤成纤维细胞，进行二维凝胶电泳，以证明蛋白质的氧化修饰，特别关注脂质过氧化的主要次级产物 4-羟基壬烯醛（4-HNE PAs）和硝基酪氨酸与蛋白质加合物的形成，这是一氧化氮（NO）或其生化相互作用的生物化学产物与活性氧（ROS）。然后，通过质谱、LC-ESI-CID-MS/MS 对氧化修饰的斑点进行鉴定。我们的结果表明，与健康对照组细胞相比，RTT 患者的成纤维细胞蛋白质组中有 15 个蛋白质斑点存在 4-HNE PAs 和/或硝基酪氨酸加合物。翻译后修饰的蛋白质与几个功能类别有关，特别是细胞骨架结构和蛋白质折叠。此外，在 RTT 成纤维细胞中观察到诱导型一氧化氮合酶（iNOS）的表达明显上调，伴有高亚硝酸盐水平，这证明了硝基酪氨酸蛋白质修饰的增加。本研究不仅描述了 RTT 成纤维细胞的蛋白质组图谱，还鉴定了 4-HNE 和硝基酪氨酸修饰的蛋白质。值得注意的是，这是首次表明 NO 途径的失调可能与 RTT 的病理生理学有关。总之，能够与 4-HNE、硝基酪氨酸或两者形成加合物的广泛蛋白质的证据证实了细胞功能的多个方面可能发生改变，这与 RTT 患者的复杂临床特征密切相关。
