title: Identification of novel interaction between ADAM17 (a disintegrin and metalloprotease 17) and thioredoxin-1.
abstract: ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.
authors.affiliation: Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Brasil.
authors.forename: Annelize Z B
authors.initials: AZ
authors.lastname: Aragão
authors.forename: Maria Luiza C
authors.initials: ML
authors.lastname: Nogueira
authors.forename: Daniela C
authors.initials: DC
authors.lastname: Granato
authors.forename: Fernando M
authors.initials: FM
authors.lastname: Simabuco
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Honorato
authors.forename: Zaira
authors.initials: Z
authors.lastname: Hoffman
authors.forename: Sami
authors.initials: S
authors.lastname: Yokoo
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
authors.forename: Fabio M
authors.initials: FM
authors.lastname: Squina
authors.forename: Ana Carolina M
authors.initials: AC
authors.lastname: Zeri
authors.forename: Paulo S L
authors.initials: PS
authors.lastname: Oliveira
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Sherman
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Paes Leme
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006367:HeLa Cells; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D011994:Recombinant Proteins; D010768:Phosphoserine; D013879:Thioredoxins; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse; D013755:Tetradecanoylphorbol Acetate