Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome

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.

在蛋白质相互作用网络中，中心蛋白通常具有大量的多样交互作用。确定这些中心蛋白的核心交互及其功能，是网络研究中的一个重大挑战。含有WD40重复序列的蛋白质代表了一类可能成为中心蛋白的大蛋白质家族。WDR76是一种表征不佳的WD40重复序列蛋白，其可能参与DNA损伤修复、细胞周期进程、细胞凋亡、基因表达调控和蛋白质质量控制等功能。WDR76具有庞大且多样的交互网络，这使得其研究变得具有挑战性。在本研究中，我们严格实施了一系列亲和纯化结合质谱分析（AP-MS），以不同生化条件下绘制WDR76的互作组。我们应用AP-MS分析结合分子排阻色谱法来解析基于WDR76的蛋白质复合物。此外，我们还展示了WDR76通过其WD40重复结构域与CCT复合物相互作用，以及与WD40结构域外的DNA-PK–KU、PARP1、GAN、SIRT1和组蛋白的相互作用。对WDR76相互作用稳定性的评估，导致了对GAN和SIRT1相互作用的聚焦和精简的反向分析，从而验证了这些相互作用。总体而言，用于研究WDR76的方法对于研究其他含有WD40重复结构域的蛋白质也将具有价值，这些结构域在许多生物体的大量蛋白质中是保守的。