Caulton, Simon
(2016)
Characterisation of the deubiquitinating enzyme USP20.
PhD thesis, University of Nottingham.
Abstract
USP20 is a deubiquitinating enzyme that is involved in a number of important cellular pathways, including thyroid metabolism, hypoxic response, seven transmembrane receptor signalling, NF-κβ signalling, centrosome homeostasis and DNA repair. Of recent, it is becoming a major deubiquitinase involved in regulating the DNA-damage response pathway and cell cycle checkpoints.
The protein consists of a zinc finger domain, catalytic domain and two ‘domain present in USP’ (DUSP) domains; an architecture shared only with its paralogue USP33. There is no structural information on any of the domains of USP20, so crystallisation trials of the domains of USP20 were performed in order to solve their structures by X-ray crystallography. In addition, yeast two-hybrid (Y2H) and in vitro assays were used to further characterise known and putative interactors of USP20. Finally, the zinc finger domain and DUSP domains were used in pull down assays to identify USP20-interacting proteins from HEK293 lysate.
Two stable and well-expressing constructs of the zing finger domain (USP20 1-101 and 1-108) were purified and set up for crystallisation trials. Buffer screens were also performed on the USP20 1-101 construct to increase its stability for crystallisation. Monodisperse, pure protein of any catalytic domain-containing construct of USP20 was unobtainable; only a trigger factor-tagged full length USP20 was purified and active. Two constructs containing the double DUSP domains were produced (USP20 686-914 and 686-894), and both suffered from a low solubility limit. Buffer screening was used to increase its stability, which identified ethylene glycol as a stabilising additive. Due to the nature of commonly used solubility tags, novel tags were designed that would potentially benefit the crystallisation of the fusion construct. Identified from the PDB and literature searches, the calponin homology domain from human β-spectrin (PDB code 1BKR) and the receiver domain from Myxococcus xanthus social motility protein frzS (PDB code 2GKG) were used. Both new tags, as well as MBP were fused to the N-terminus of the DUSP domains (USP20 686-894) to enhance solubility and crystallise the DUSP domains. 2GKG was an effective solubility tag, increased the solubility of the DUSP domains to near that of the MBP fusion. 1BKR, however, was only marginally useful as a solubility tag. In total 97 crystallisation trials were set up for all constructs of USP20, but no crystals containing USP20 protein formed.
Y2H assays were used to investigate the interaction between USP20 domains and Β-arrestin-1, TRAF6, RAD17 and PLK1. Of these, only and interaction between USP20’s DUSP domains (residues 686-894) and full length PLK1 was observed. Interestingly, further Y2H and ELISA showed a non-canonical, binary interaction between the poloboxes of PLK1 (residues 367-603) and the DUSP domains. Pull down assays produced a list of possible novel interactors for USP20. These include proteins implicated in processes known, and unknown, to involve USP20. Finally, using ELISA, thermal shift assays and ITC, it was shown that the zinc finger domain of USP20 does not bind to ubiquitin.
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