title: Enrichment of Metabolite-Binding Proteins by Affinity Elution in Tandem Hydrophobic Interaction Chromatography (AETHIC) Reveals RKIP Regulating ERK Signaling in an ATP-Dependent Manner.
abstract: To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1, resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP's inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.
authors.affiliation: Graduate Institute of Biochemical Sciences, National Taiwan University , No.1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Wei-Chieh
authors.initials: WC
authors.lastname: Huang
authors.affiliation: Graduate Institute of Integrated Medicine, China Medical University , No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan.
authors.forename: Der-Yen
authors.initials: DY
authors.lastname: Lee
authors.affiliation: Graduate Institute of Biochemical Sciences, National Taiwan University , No.1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Geen-Dong
authors.initials: GD
authors.lastname: Chang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D020935:MAP Kinase Signaling System; D049488:Phosphatidylethanolamine Binding Protein / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013552:Swine
chemical_list: D002352:Carrier Proteins; D008024:Ligands; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D000255:Adenosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf
keywords: ATP; RKIP; Raf-1; glucose depletion; hydrophobic interaction; metabolite