半胱氨酸介导的细胞信号转导在纤维连接蛋白片段刺激的软骨细胞中的氧化还原调节。
Cysteine-Mediated Redox Regulation of Cell Signaling in Chondrocytes Stimulated With Fibronectin Fragments.
机构信息
University of North Carolina School of Medicine, Chapel Hill.
Wake Forest University School of Medicine, Winston-Salem, North Carolina.
出版信息
Arthritis Rheumatol. 2016 Jan;68(1):117-26. doi: 10.1002/art.39326.
OBJECTIVE
Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling.
METHODS
Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor.
RESULTS
Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f-induced production of matrix metalloproteinase 13.
CONCLUSION
These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation.
目的
细胞内蛋白质的氧化后翻译修饰可能会调节与关节炎中软骨破坏相关的信号通路。在这项研究中，我们研究了骨关节炎（OA）软骨细胞中半胱氨酸残基氧化形成亚磺酸（S-亚磺酰化）的情况，并探讨了纤维连接蛋白（FN-f）片段刺激软骨细胞分解代谢信号的机制。
方法
使用专门且不可逆地与蛋白质 S-亚磺酰化半胱氨酸反应的二甲基酮类似物分析来自 OA 和正常人类关节软骨的软骨细胞。通过免疫印迹法在有和没有 FN-f 刺激的情况下测量细胞裂解物中的总 S-亚磺酰化水平，并通过共聚焦显微镜在固定细胞中测量。通过质谱法鉴定特定蛋白质中的 S-亚磺酰化，并通过免疫印迹法进行验证。使用荧光共振能量转移生物传感器在活细胞中测量Src 活性。
结果
与正常软骨细胞中的 S-亚磺酰化水平相比，来自 OA 软骨的软骨细胞中的蛋白质发现具有更高的基础 S-亚磺酰化水平。用 FN-f 处理正常软骨细胞会诱导多种蛋白质（包括酪氨酸激酶 Src）的 S-亚磺酰化水平升高。FN-f 处理还增加了 Src 活性水平。用二甲基酮预处理以改变 S-亚磺酰化功能或用 Src 激酶抑制剂预处理可抑制 FN-f 诱导的基质金属蛋白酶 13 的产生。
结论
这些结果首次证明了 S-亚磺酰化在人类关节软骨细胞中蛋白质的氧化后翻译修饰的存在。由于能够调节包括纤维连接蛋白片段诱导的分解代谢介质在内的许多细胞信号通路的活性，S-亚磺酰化可能导致 OA 中的软骨破坏，值得进一步研究。
Zotero 插件