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Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry

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Title: Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry
Authors: Rega, C
Russo, R
Focà, A
Sandomenico, A
Iaccarino, E
Raimondo, D
Milanetti, E
Tornatore, L
Franzoso, G
Pedone, PV
Ruvo, M
Chambery, A
Item Type: Journal Article
Abstract: GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.
Issue Date: 20-Mar-2018
Date of Acceptance: 19-Mar-2018
URI: http://hdl.handle.net/10044/1/61425
DOI: https://dx.doi.org/10.1016/j.ijbiomac.2018.03.090
ISSN: 1879-0003
Publisher: Elsevier
Start Page: 114
End Page: 123
Journal / Book Title: International Journal of Biological Macromolecules
Volume: 114
Replaces: http://hdl.handle.net/10044/1/58432
10044/1/58432
Copyright Statement: © 2018 The Authors. Published by Elsevier B.V. This is an open access article under a CC-BY Attribution Licence (https://creativecommons.org/licenses/by/4.0/)
Sponsor/Funder: Medical Research Council (MRC)
Medical Research Council (MRC)
Cancer Research UK
Funder's Grant Number: G0901436
MR/L005069/1
15115
Keywords: Chemical cross-linking
GADD45β
MKK7
Mass spectrometry
Protein-protein interaction
0601 Biochemistry And Cell Biology
Polymers
Publication Status: Published
Appears in Collections:Department of Medicine
Faculty of Medicine



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