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Interactome of ANKRD55, an autoimmune disease risk protein with unknown function

frontiersin. 2019;

Nerea Ugidos, †, Jorge Mena, †, Sara Baquero, Iraide Alloza, Mikel Azkargorta, Felix Elortza and Koen Vandenbroeck, *

Products/Services Used	Details	Operation
Proteins, Expression, Isolation and Analysis	The supernatant was carefully removed, and nuclei were lysed with RIPA buffer. For the IP of ANKRD55 isoforms together with their binding partners, ANKRD55 and control samples were incubated with Anti-DDK G1 mouse monoclonal antibody coupled to the resin (cat.no. L00432; Genscript) at 4°C overnight (o/n), on rotating wheel. After incubation, resin was washed six times with washing buffer containing 50 mM Tris-HCl and 150 mM NaCl, pH adjusted to 7.4 (phosphatase inhibitors were included for interactome analysis) and ANKRD55 001 complexes were eluted using acidic elution buffer (0.1 M glycine, 0.15 M NaCl and 0,5% SDS pH 2).	Get A Quote

摘要

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4+ T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, β-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.