Ribosomal protein S3: A multi-functional protein that interacts with both p53 and MDM2 through its KH domain.
Ribosomal protein S3: A multi-functional protein that interacts with both p53 and MDM2 through its KH domain.
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DOI：
10.1016/j.dnarep.2009.07.003
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发表时间：
2009-10-02
期刊：
影响因子：
3.8
通讯作者：
Deutsch WA
中科院分区：
文献类型：
--
作者：
Yadavilli S;Mayo LD;Higgins M;Lain S;Hegde V;Deutsch WA
p53蛋白响应细胞应激，调节参与细胞周期、细胞凋亡和DNA修复的基因。在正常情况下，p53水平通过mdm2介导的泛素化和蛋白体降解保持在较低水平。为了寻找参与这一调控环的新蛋白，我们进行了MDM2肽下拉分析和质谱分析，以筛选MDM2的潜在相互作用伙伴。我们鉴定了核糖体蛋白S3 (RPS3)，通过His和GST下拉实验、荧光共振能量转移和原位接近结扎实验进一步确定了它与MDM2，尤其是p53的相互作用。此外，在暴露于氧化应激的细胞中，p53水平在24小时内略有上升，而MDM2水平在暴露6小时后下降，但在接下来的18小时内上升。相反，在暴露于氧化应激并携带siRNA敲低RPS3表达的细胞中，观察到p53水平下降，MDM2所拥有的E3泛素连接酶结构域丢失。使用7,8 -二氢-8-氧鸟嘌呤双寡核苷酸作为底物的DNA pull-down实验发现，RPS3作为MDM2和p53额外结合的支架，表明RPS3与维持基因组完整性的重要蛋白质相互作用。
The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 hrs, whereas MDM2 levels declined after 6 hrs exposure, but rose over the next 18 hrs of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 was observed. DNA pull-down assays using a 7, 8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.
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