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，中心蛋白往往具有大量多样化的相互作用。确定其核心相互作用及其功能，对于网络研究而言，构成一项重大挑战。具有WD40重复序列的蛋白质构成了一类庞大的蛋白质家族，它们可能充当中心蛋白。WDR76是一种特性描述不充分的WD40重复序列蛋白，可能与DNA损伤修复、细胞周期进程、细胞凋亡、基因表达调控以及蛋白质质量控制等功能相关。WDR76拥有庞大而多样化的相互作用网络，这使得其研究变得尤为艰巨。在本研究中，我们严格实施了亲和纯化结合质谱分析（AP-MS）的一系列分析，以不同生化条件下绘制WDR76的相互作用图谱。我们应用了AP-MS分析结合排阻色谱技术，解析以WDR76为基础的蛋白质复合物。此外，我们还展示了WDR76通过其WD40重复序列域与CCT复合物相互作用，以及与WD40域以外的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，导致了一系列针对性的逆向分析，验证了与GAN和SIRT1的相互作用。总体而言，用于研究WDR76的方法对于研究其他含有WD40重复序列域的蛋白质具有重要价值，这些序列域在众多生物体中的大量蛋白质中均得以保守。