10.1021/acs.jproteome.9b00373.s001 Gerald Dayebgadoh Gerald Dayebgadoh Mihaela E. Sardiu Mihaela E. Sardiu Laurence Florens Laurence Florens Michael P. Washburn Michael P. Washburn Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome American Chemical Society 2019 CCT PARP WDR 76 DNA-PK WDR 76 interactome WD 40 domain gene expression regulation protein interaction networks study WDR 76 Diverse WDR 76 Protein Interactome SIRT WD 40 hub protein protein quality control DNA damage repair WDR 76-based protein complexes GAN AP WDR 76 interactions 2019-08-09 13:41:42 Journal contribution https://acs.figshare.com/articles/journal_contribution/Biochemical_Reduction_of_the_Topology_of_the_Diverse_WDR76_Protein_Interactome/9441713 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.