Interactions and Functions of the Ubiquitin Specific Protease 7 in Human Cells

The ubiquitin-specific protease 7 (USP7) is a deubiquitylating enzyme that regulates many important cellular processes and is highly studied for its contributions to cancer. Our lab has previously discovered a binding pocket in the USP7 N-terminal TRAF domain that mediates interactions with target proteins. In this thesis I demonstrate how I contributed to the identification of a second binding pocket located in the Ubl2 ubiquitin-like structure of the USP7 C-terminal domain. Furthermore, to gain a more comprehensive understanding of the interactions and functions of USP7, I used a proteomics approach to profile USP7 interactions in cancer cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 and identified novel interactions with FBXO38 and two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. I show that USP11, PPM1G, DDX24, TRIP12 and FBXO38 bind USP7 through its TRAF binding pocket, while DHX40 interacts through the Ubl2 pocket. Motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity showed that USP7 consistently stabilizes DDX24, DHX40, TRIP12 and FBXO38 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Together these results better define the mechanisms of USP7 interactions and identify FBXO38, DDX24, and DHX40 as new USP7 targets. Furthermore, a BioID approach was used to profile the interactions and putative functions of FBXO38, revealing an interaction with KIF20B, a Kinesin-6 protein required for cytokinesis. I show that depletion of either FBXO38 or USP7 result in dramatic decreases in total KIF20B levels and its localization to the midbody, which were manifested in cytokinetic defects. Furthermore, cytokinetic defects associated with USP7 silencing were rescued by restoring FBXO38 or KIF20B. Therefore, I have identified novel roles for USP7 and FBXO38 in the regulation of cytokinesis.

Department dc:contributor.department

Molecular and Medical Genetics

Year dc:date.issued

2019

Author dc:creator

• Georges, Anna-Marie

Advisor dc:contributor.advisor

• Frappier, Lori

• Deubiquitylation
• Interactions
• proteomics
• Ubiquitin
• USP7

Handle dc:identifier.uri

http://hdl.handle.net/1807/95849

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/95849