Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, Rome, Italy.
Biotechnol Adv. 2012 Jan-Feb;30(1):4-15. doi: 10.1016/j.biotechadv.2011.06.012. Epub 2011 Jun 29.
Families of conserved protein domains, specialized in mediating interactions with short linear peptide motifs, are responsible for the formation of a variety of dynamic complexes in the cell. An important subclass of these motifs are characterized by a high proline content and play a pivotal role in biological processes requiring the coordinated assembly of multi-protein complexes. This is achieved via interaction of proteins containing modules such as Src Homology-3 (SH3) or WW domains and specific proline rich patterns. Here we make available via a publicly accessible database a synopsis of our current understanding of the interaction landscape of the human SH3 protein family. This is achieved by integrating an information extraction strategy with a new experimental approach. In a first approach we have used a text mining strategy to capture a large number of manuscripts reporting interactions between SH3 domains and target peptides. Relevant information was annotated in the MINT database. In a second experimental approach we have used a variant of the WISE (Whole Interactome Scanning Experiment) strategy to probe a large number of naturally occurring and chemically-synthesized peptides arrayed at high density on a glass surface. By this method we have tested 60 human SH3 domains for their ability to bind a collection of 9192 poly-proline containing peptides immobilized on a glass chip. To evaluate the quality of the resulting interaction dataset, we retested some of the interactions on a smaller scale and performed a series of pull down experiments on native proteins. Peptide chips, pull down assays, SPOT synthesis and phage display experiments have allowed us to further characterize the specificity and promiscuity of proline-rich binding domains and to map their interaction network. Both the information captured from the literature and the interactions inferred from the peptide chip experiments were collected and stored in the PepspotDB (http://mint.bio.uniroma2.it/PepspotDB/).
保守蛋白结构域家族，专门介导与短线性肽基序的相互作用，负责在细胞中形成各种动态复合物。这些基序的一个重要亚类的特点是脯氨酸含量高，在需要多蛋白复合物协调组装的生物过程中起着关键作用。这是通过含有 Src Homology-3 (SH3) 或 WW 结构域等模块的蛋白质与特定富含脯氨酸的模式之间的相互作用来实现的。在这里，我们通过一个公共可访问的数据库提供了对人类 SH3 蛋白家族相互作用景观的当前理解的概述。这是通过整合信息提取策略和新的实验方法来实现的。在第一种方法中，我们使用了一种文本挖掘策略来捕获大量报道 SH3 结构域与靶肽之间相互作用的文献。相关信息在 MINT 数据库中进行了注释。在第二种实验方法中，我们使用了 WISE（Whole Interactome Scanning Experiment）策略的变体来探测大量自然发生和化学合成的肽，这些肽以高密度排列在玻璃表面上。通过这种方法，我们测试了 60 个人类 SH3 结构域与固定在玻璃芯片上的 9192 个富含脯氨酸的肽集合的结合能力。为了评估得到的相互作用数据集的质量，我们在较小的规模上重新测试了一些相互作用，并在天然蛋白质上进行了一系列下拉实验。肽芯片、下拉实验、SPOT 合成和噬菌体展示实验使我们能够进一步表征富含脯氨酸的结合结构域的特异性和混杂性，并绘制其相互作用网络。从文献中捕获的信息和从肽芯片实验中推断出的相互作用都被收集并存储在 PepspotDB（http://mint.bio.uniroma2.it/PepspotDB/）中。
