Quantitative protein network monitoring in response to DNA damage
Quantitative protein network monitoring in response to DNA damage
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DOI：
10.1021/pr0702971
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发表时间：
2008-02-01
影响因子：
4.4
通讯作者：
Austin, John
中科院分区：
文献类型：
--
作者：
Nishizuka, Satoshi;Ramalingam, Sundhar;Austin, John
传统的分子生物学技术已经确定了大量的细胞信号传导途径;然而，这些途径的重要性往往会有所不同，这取决于治疗类型，剂量，治疗后的时间和细胞类型等因素。在这里，我们描述了一种技术，使用“反相”蛋白裂解物微阵列。（RPA）来获得关于蛋白质动态响应DNA损伤的多个维度的信息。从三种细胞应激处理（IR、UV和ADR）中收集全细胞裂解物，每次处理4个剂量，每个剂量依次在10个时间点，得到由10 240个特征组成的单载玻片RPA，包括重复。通过相应强制降解条件下的FACS分析，将18种独特蛋白质种类的动态分子谱与表型命运进行比较。我们在这个新平台上的初步定量结果证实：（1）p53蛋白存在明显的应激剂量反应效应;（2）p21和Cyclin D3/p53-Ser 15对DNA损伤的反应性增加速率的比较可能与DNA含量的模式有关。这种方法，提供了一个定量的蛋白质表达水平的时程监测，可以为开发细胞信号动力学的数学模型提供实验参考。虽然目前的研究主要集中在DNA损伤修复途径，但该技术通常适用于蛋白质信号转导的研究。
Conventional molecular biology techniques have identified a large number of cell signaling pathways; however, the importance of these pathways often varies, depending on factors such as treatment type, dose, time after treatment, and cell type. Here, we describe a technique using "reverse-phase" protein lysate microarrays. (RPAs) to acquire multiple dimensions of information on protein dynamics in response to DNA damage. Whole-cell lysates from three cellular stress treatments (IR, UV, and ADR) were collected at four doses per treatment, and each, in turn, at 10 time points, resulting in a single-slide RPA consisting of 10 240 features, including replicates. The dynamic molecular profile of 18 unique protein species was compared to phenotypic fate by FACS analysis for corresponding stress conditions. Our initial quantitative results in this new platform confirmed that (1) there is clear stress dose-response effect in p53 protein and (2) a comparison of the rates of increase of p21 and Cyclin D3/p53-Ser15 in response to DNA damage may be associated with the pattern of DNA content. This method, offering a quantitative time-course monitoring of protein expression levels, can provide an experimental reference for developing mathematical models of cell signaling dynamics. Although the present study focuses on the DNA damage-repair pathway, the technique is generally useful to the study of protein signaling..