Title
Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase : suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2) Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase : suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2)
Author
Faculty/Department
Faculty of Sciences. Chemistry
Publication type
article
Publication
Baltimore, Md ,
Subject
Chemistry
Biology
Source (journal)
Journal of biological chemistry. - Baltimore, Md
Volume/pages
290(2015) :7 , p. 4178-4191
ISSN
0021-9258
ISI
000349458400029
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5SOCS2 complexes is supported by traveling wave ion mobility mass spectrometry data.
E-info
https://repository.uantwerpen.be/docman/iruaauth/72ee10/c4d11c63260.pdf
Full text (open access)
https://repository.uantwerpen.be/docman/irua/521d09/9862.pdf
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