title: Purification of the COP9 signalosome complex and binding partners from human T cells.
abstract: The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral-host interactions or immune activation in their natural milieu.
authors.affiliation: Department of Biology, San Diego State University, San Diego, California, USA.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Stotland
authors.forename: Laura
authors.initials: L
authors.lastname: Pruitt
authors.forename: Phillip
authors.initials: P
authors.lastname: Webster
authors.forename: Roland
authors.initials: R
authors.lastname: Wolkowicz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D000075686:COP9 Signalosome Complex; D002853:Chromatography, Liquid; D017931:DNA Primers; D005434:Flow Cytometry; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D025581:Neurofibromin 2 / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D046912:Multiprotein Complexes; D025581:Neurofibromin 2; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex