Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

Autor(es):Mostrar menos -

Aragao, Annelize Z. B. ^[1] ; Nogueira, Maria Luiza C. ^[1] ; Granato, Daniela C. ^[1] ; Simabuco, Fernando M. ^[1] ; Honorato, Rodrigo V. ^[1] ; Hoffman, Zaira ^[2] ; Yokoo, Sami ^[1] ; Laurindo, Francisco R. M. ^[3] ; Squina, Fabio M. ^[2] ; Zeri, Ana Carolina M. ^[1] ; Oliveira, Paulo S. L. ^[1] ; Sherman, Nicholas E. ^[4] ; Paes Leme, Adriana F. ^[1]

Afiliação do(s) autor(es)

^[1] CNPEM, Lab Espectrometria Massas, Lab Nacl Biociencias, LNBio, BR-13083970 Campinas, SP - Brazil

^[2] CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol, CTBE, BR-13083970 Campinas, SP - Brazil

^[3] Univ Sao Paulo, Inst Coracao, BR-05403000 Sao Paulo - Brazil

^[4] Univ Virginia, WM Keck Biomed Mass Spectrometry Lab, Charlottesville, VA 22908 - USA

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biological Chemistry; v. 287, n. 51, p. 43071-43082, DEC 14 2012.
• Citações Web of Science: 22

Resumo

ADAM17, which is also known as TNF alpha-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. (AU)

• Processo FAPESP: 11/02267-9 - Busca de ligantes e Mapeamento dos domínios da ADAM-17 responsáveis pela interação com os ligantes.
• Beneficiário: Daniela Campos Granato
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 09/54067-3 - EMU: aquisição de um espectrômetro de massas acoplado a cromatografia líquida para permitir ampliar a capacidade de atendimento de usuários e disponibilizar novas tecnologias no Laboratório de Espectrometria de Massas do Centro de Biologia Molecular Estrutural (ABTLUS)
• Beneficiário: Adriana Franco Paes Leme
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários
• Processo FAPESP: 10/19278-0 - Estudo da regulação de ADAMs em câncer oral
• Beneficiário: Adriana Franco Paes Leme
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 10/15675-5 - Efeito da modulação da metaloproteinase de membrana ADAM-17 no subproteoma extracelular utilizando modelo de câncer oral
• Beneficiário: Fernando Moreira Simabuco
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 09/18301-1 - Estudo da regulação da ADAM17 recombinante pela determinação dos sítios de fosforilação do domínio citoplasmático e de seus ligantes em células normais e de câncer.
• Beneficiário: Annelize Zambon Barbosa Aragão
• Modalidade de apoio: Bolsas no Brasil - Doutorado