title: Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase.
abstract: Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.
authors.affiliation: Laboratories of Lymphocyte Biology, The Rockefeller University, New York, NY 10065, USA. rdelker@rockefeller.edu
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Delker
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Zhou
authors.forename: Alexandros
authors.initials: A
authors.lastname: Strikoudis
authors.forename: C Erec
authors.initials: CE
authors.lastname: Stebbins
authors.forename: F Nina
authors.initials: FN
authors.lastname: Papavasiliou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D017124:Conserved Sequence; D003564:Cytidine Deaminase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D054829:RING Finger Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054875:Ubiquitination; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D011994:Recombinant Proteins; C578829:RNF126 protein, human; D044767:Ubiquitin-Protein Ligases; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase