Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity

Author(s):Show less -

Granato, Daniela C. ^[1] ; P. e Costa, Rute A. ^[1] ; Kawahara, Rebeca ^[1] ; Yokoo, Sami ^[1] ; Aragao, Annelize Z. ^[1] ; Domingues, Romenia R. ^[1] ; Pauletti, Bianca A. ^[1] ; Honorato, Rodrigo V. ^[1] ; Fattori, Juliana ^[1] ; Figueira, Ana Carolina M. ^[1] ; Oliveira, Paulo S. L. ^[1] ; Consonni, Silvio R. ^[1] ; Fernandes, Denise ^[2] ; Laurindo, Francisco ^[2] ; Hansen, Hinrich P. ^[3] ; Leme, Adriana F. Paes ^[1]

Affiliation

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

^[2] Univ Sao Paulo, Fac Med, Inst Coracao, Sao Paulo - Brazil

^[3] Univ Hosp Cologne, Dept Internal Med 1, Cologne - Germany

• Document type: Journal article
• Source: Antioxidants & Redox Signaling; v. 29, n. 8 FEB 2018.
• Web of Science Citations: 0

Abstract

Aims: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. Results: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1(K72A) and catalytic site mutant Trx-1(C32/35S) rescued ADAM17 activity, although the interaction with Trx-1(C32/35S) was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1(C32/35S) mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1(K72A) mutant showed similar oxidant levels to Trx-1(C32/35S), even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. Innovation: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. Conclusion: This unexpected Trx-1(K72A) behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. (AU)

• FAPESP's process: 16/01528-7 - The effect of Trx-1 overexpression on protein expression and redox state of cysteines by iodoTMT and TMT labeling
• Grantee: Rute Alves Pereira e Costa
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 09/54067-3 - Acquisition of a mass spectrometer coupled to a liquid chromatography system for increasing the capacity to meet the needs of users and for making new technologies available in the Laboratory of Mass Spectrometry
• Grantee: Adriana Franco Paes Leme
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 14/06485-9 - Investigation of the ADAM17 metalloproteinase regulation mechanism by the interaction with Trx-1
• Grantee: Rute Alves Pereira e Costa
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/23888-0 - Studies on the activation mechanisms of MMP-2 and ADAM17: identification of regulatory proteins, oxidant production pathways, epigenetics and proteolytic targets
• Grantee: Raquel Fernanda Gerlach
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 11/02267-9 - Identifying ADAM17 partners and mapping the domains responsible for its interaction with other proteins.
• Grantee: Daniela Campos Granato
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 10/19278-0 - Study of regulation of ADAMs in oral cancer
• Grantee: Adriana Franco Paes Leme
• Support Opportunities: Research Grants - Young Investigators Grants