Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome
Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome
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DOI：
10.1021/acs.jproteome.9b00373
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发表时间：
2019-09-01
影响因子：
4.4
通讯作者：
Washburn, Michael P.
中科院分区：
文献类型：
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作者：
Dayebgadoh, Gerald;Sardiu, Mihaela E.;Washburn, Michael P.
蛋白质相互作用网络中的中心蛋白质通常具有大量不同的相互作用。确定这种中心蛋白的核心相互作用和功能仍然是网络研究中的重大挑战。具有 WD40 重复序列的蛋白质代表一大类可以作为枢纽蛋白的蛋白质。 WDR76 是一种尚未充分表征的 WD40 重复蛋白，可能参与 DNA 损伤修复、细胞周期进展、细胞凋亡、基因表达调控和蛋白质质量控制。 WDR76 拥有庞大且多样化的交互网络，这使其研究具有挑战性。在这里，我们严格执行一系列亲和纯化与质谱（AP-MS）分析相结合，通过不同的生化条件绘制出 WDR76 相互作用组。我们应用 AP-MS 分析与尺寸排阻色谱相结合来解析基于 WDR76 的蛋白质复合物。此外，我们还表明，WDR76 通过其 WD40 重复结构域与 CCT 复合物相互作用，并与 WD40 结构域之外的 DNA-PK-KU、PARP1、GAN、SIRT1 和组蛋白相互作用。对 WDR76 相互作用稳定性的评估导致了集中且简化的相互分析，验证了与 GAN 和 SIRT1 的相互作用。总体而言，用于研究 WDR76 的方法对于研究含有 WD40 重复结构域的其他蛋白质很有价值，这些重复结构域在许多生物体的大量蛋白质中是保守的。
A hub protein in protein interaction networks will typically have a large number of diverse interactions. Determining the core interactions and the function of such a hub protein remains a significant challenge in the study of networks. Proteins with WD40 repeats represent a large class of proteins that can be hub proteins. WDR76 is a poorly characterized WD40 repeat protein with possible involvement in DNA damage repair, cell-cycle progression, apoptosis, gene expression regulation, and protein quality control. WDR76 has a large and diverse interaction network that has made its study challenging. Here we rigorously carry out a series of affinity purification coupled to mass spectrometry (AP-MS) analyses to map out the WDR76 interactome through different biochemical conditions. We apply AP-MS analysis coupled to size-exclusion chromatography to resolve WDR76-based protein complexes. Furthermore, we also show that WDR76 interacts with the CCT complex via its WD40 repeat domain and with DNA-PK-KU, PARP1, GAN, SIRT1, and histones outside of the WD40 domain. An evaluation of the stability of WDR76 interactions led to focused and streamlined reciprocal analyses that validate the interactions with GAN and SIRT1. Overall, the approaches used to study WDR76 would be valuable to study other proteins containing WD40 repeat domains, which are conserved in a large number of proteins in many organisms.