Analysis of the STAT3 interactome using in-situ biotinylation and SILAC
Analysis of the STAT3 interactome using in-situ biotinylation and SILAC
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DOI：
10.1016/j.jprot.2013.08.021
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发表时间：
2013-12-06
影响因子：
3.3
通讯作者：
Horn, Friedernann
中科院分区：
文献类型：
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作者：
Blumert, Conny;Kalkhof, Stefan;Horn, Friedernann
信号转导子和转录激活子3（STAT 3）被多种细胞因子和生长因子激活。为了生成与STAT 3特异性相互作用的蛋白质的全面数据集，我们在细胞培养物中应用了氨基酸稳定同位素标记（SILAC）。对于使用链霉亲和素的高亲和力下拉，我们将STAT 3与允许原位生物素化的短肽标签（生物标签）融合，这不影响STAT 3功能。通过这种方法，在人胚肾-293细胞中检测到3642个共沉淀蛋白。使用统计和功能标准过滤最终提取了136个蛋白质作为STAT 3的推定相互作用伴侣。物理相互作用网络分析和已知和预测的相互作用伙伴的富集表明，我们的过滤标准成功地富集了真正的STAT 3相互作用。我们的方法确定了许多新的相互作用，包括以前预测与STAT 3相关的相互作用。通过相互共沉淀，我们能够验证STAT 3和选定的相互作用物之间的物理关联，包括与TOX高迁移率族盒家族成员TOX 4的新型相互作用。应用相同的方法，我们接下来研究了STAT 3相互作用组的激活依赖性。我们再次确定了已知和新的相互作用。生物学意义蛋白质的定位、活性、功能、降解和合成受蛋白质与其他蛋白质、生物大分子和小分子的相互作用的调节。因此，在给定的蛋白质组中蛋白质相互作用的综合表征是理解细胞生物化学的下一个里程碑。为了生成与所选诱饵蛋白特异性相互作用的蛋白质的全面的相互作用组数据集，我们将我们的诱饵蛋白STAT 3与允许原位生物素化的短肽标签（生物标签）融合。这种生物标签允许使用链霉亲和素的亲和下拉，但既不影响STAT 3通过酪氨酸磷酸化的激活，也不影响其反式激活潜力。我们结合SILAC的精确相对蛋白定量、亚细胞分级以增加相互作用蛋白的覆盖率、高亲和力pull-down和严格的过滤方法成功地分析了STAT 3的相互作用物组。该方法为研究蛋白质相互作用及其对翻译后修饰的依赖性提供了一种快速有效的方法。(C)2013年由Elsevier B. V.出版
Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein protein interaction effectively and comprehensively.Biological significanceThe location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3.With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications. (C) 2013 Published by Elsevier B.V.