Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes
Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes
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DOI：
10.1073/pnas.1312296111
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发表时间：
2014-02-18
影响因子：
11.1
通讯作者：
Kim, Philip M.
中科院分区：
文献类型：
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作者：
Ivarsson, Ylva;Arnold, Roland;Kim, Philip M.
人类蛋白质组包含大量的短线性基序（SLiM），其充当模块化蛋白质结构域的结合界面。这种相互作用对于信号传导和其他细胞过程至关重要，但由于它们的低至中等亲和力而难以检测。在这里，我们开发了一种专门的方法，即蛋白质组肽-噬菌体展示（ProP-PD），来鉴定结构域与SLiM的相互作用。具体而言，我们使用定制的寡核苷酸微阵列产生了含有所有人类和病毒C-末端肽的噬菌体文库。利用这些文库，我们筛选了人Densin-180、Erbin、Scribble和大分子同源物1的9个PSD-95/Dlg/ZO-1（PDZ）结构域的肽配体。我们确定了几个已知的和假定的相互作用可能与细胞信号转导途径，并确认全长Scribble和靶蛋白β-PIX，plakophilin-4，鸟苷酸环化酶可溶性亚基α-2使用共定位和免疫共沉淀实验之间的相互作用。重组Scribble PDZ结构域和代表这些蛋白质的C末端的合成肽的亲和力在1至40 μ M范围内。此外，我们确定了几个公认的宿主-病毒蛋白质-蛋白质相互作用，并证实了PDZ结构域的Scribble与人T细胞白血病病毒的Tax-1的C末端的微摩尔亲和力。以前未知的假定病毒蛋白配体的PDZ结构域的Scribble和Erbin也被确定。因此，我们证明了我们的ProP-PD库是探测PDZ结构域相互作用的有用工具。该方法可以扩展到询问所有潜在的真核生物，细菌和病毒的SLiMs，我们认为这将是一个非常有价值的方法，用于研究细胞和病原体-宿主蛋白质-蛋白质相互作用。
The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins beta-PIX, plakophilin-4, and guanylate cyclase soluble subunit alpha-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-mu M range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.