In vitro studies on the recognition of NF-κB p65 subunit by the deubiquitinase enzyme USP7

Q Science (General);QR180 Immunology

Mitxitorena, Izaskun ; Carmody, Ruaidhrí J.

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: NF-κB, p65, USP7, ubiquitination, interaction, structure.;   

Others  :  http://theses.gla.ac.uk/40917/1/2018MitxitorenaPhD.pdf

来源: University of Glasgow

【 摘 要 】

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family plays a key role in the regulation of the immune response and the transcriptional response to infection through transcriptional activation of genes involved in those processes. The NF-κB response is regulated in the nucleus by the balance between ubiquitination and deubiquitination processes. Ubiquitination of the p65 subunit of NF-κB terminates the NF-κB response by targeting p65 for proteasomal degradation. Nevertheless, the ubiquitin molecules can be removed from targeted proteins by the action of deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme from the ubiquitin-specific protease (USP) family which deubiquitinates p65. Besides p65 deubiquitination, USP7 is involved in a huge variety of biological processes due to stabilisation or localisation of proteins involved in those processes. USP7 is a multidomain protein formed by an N-terminal Meprin and tumour necrosis factor receptor-associated factor homology (MATH) / tumour necrosis factor receptor-associated factor (TRAF) domain, a catalytic domain (CD) and five ubiquitin-like domains (UBLs) in the C-terminal region. p65 recognition by USP7 takes place through the C-terminal region, but the molecular determinants involved in the interaction are still unknown. New therapeutic compound design strategies are based on interrupting the interaction interface between both proteins involved in the interaction. Therefore, in order to design a specific inhibitor of the deubiquitinase activity of USP7 on p65 we performed a peptide array and subsequent alanine scan followed by site directed mutagenesis experiments. We concluded that UBL2 of USP7 is necessary for the interaction with p65. UBL2 deletion completely abolishes the interaction and the deubiquitinase activity of USP7 on p65. Specificity of this mutant was tested by immunoprecipitation assays with different USP7 substrates. In silico modelling revealed a putative binding pocket in USP7 UBL2 that may be targeted to inhibit the interaction with p65. Together our data suggest that a binding pocket present on UBL2 composed by amino-acids 627-ARSNGTK-633, 736-EEVKPNLTER-745 and 757-LDELMDGD-764 directs the interaction with p65, besides UBL2 when deleted inhibits the interaction with p65 and subsequently its deubiquitination in a substrate specific manner.