title: Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5.
abstract: NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5.
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: 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: 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: Xujing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Eguchi
authors.affiliation: Systems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda Maryland; and.
authors.forename: Shouguo
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Mukoyama
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: D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C541075:CHTOP protein, human; C118125:NFAT5 protein, human; C440251:NUP160 protein, human; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C000609717:Nup205 protein, human; D010455:Peptides; D011506:Proteins; D014157:Transcription Factors
keywords: intrinsic disorder; osmolality; proteomics