title: Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5.
abstract: NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Jenna F
authors.initials: JF
authors.lastname: Dumond
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.|Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Izumi
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Kevin
authors.initials: K
authors.lastname: Ramkissoon
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Marjan
authors.initials: M
authors.lastname: Gucek
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Xujing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and Maurice_burg@nih.gov.
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, Maryland; and.
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D050778:NFATC Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D009995:Osmosis; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D060066:Protein Interaction Maps; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D002457:Cell Extracts; D050778:NFATC Transcription Factors; D010455:Peptides; D020033:Protein Isoforms; D012965:Sodium Chloride
keywords: NFAT5; disorder; hypertonicity; peptide affinity; proteomics