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Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome.

ABSTRACT: 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.

SUBMITTER: Dayebgadoh G

PROVIDER: S-EPMC7295179 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome.

Dayebgadoh Gerald G Sardiu Mihaela E ME Florens Laurence L Washburn Michael P MP

Journal of proteome research 20190809 9

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 ...[more]

PMID: 31353912

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Project description:Herein, we focus on native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), and illustrate its utility in the characterization of protein complex topology and glycoprotein heterogeneity. ECCR is illustrated to effectively spread the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurement out to greater than 100,000 m/z. For both the pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) lowers the charge states and produces more topologically informative fragmentation. While more native-like fragmentation has previously been illustrated for complexes charge reduced by proton abstraction in solution, this is the first illustration that ECCR can lead to more native-like SID fragmentation of protein complexes. Application to protein glycosylation, one of the most common and diverse protein posttranslational modifications, is also illustrated because glycosylation is important for structural and functional properties and plays essential roles in many key biological processes. The immense heterogeneity resulting from variability in glycosylation sites and glycan composition and structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Data for stabilized heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer illustrate that ECCR enables significantly improved resolution of glycan heterogeneity. Without ECCR, the charge states of a glycoprotein complex are not resolved and average mass determination is available only through the use of charge detection mass spectrometry or mass photometry. With ECCR after narrow m/z selection, multiple glycoform m/z values are apparent, providing quick global, glycoform profiling and providing a future path for glycan localization on individual intact glycoforms (e.g., though top-down dissociation).

Dataset Information

Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome.

Publications

Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome.

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