Structure And Function Studies Of Deubiquitinase Usp28

Objective: Usp28(Ubiquitin-specific protease28), which is one of the newestanti-cancer drug targets of the deubiquitinase family, plays key roles in the occurringand the developing of certain types of cancers by controlling the stabilities of theoncoproteins MYC (Myelocytomatosis virus oncoprotein) and LSD1(Lysine-specific demethylase1). More interestingly, the bioinformatic analysis dataindicates that there is a high possibility that the function of Usp28is cross-regulatedby ubiquitylation and another kind of protein post-translational modification–SUMOylation. The NMR-based research work, which is carried out and presented inthis thesis, confirms that the cross-regulation truly exists and also simultaneouslyelucidates the working mechanism and structural basis of the regulation in molecularlevel. The results that we have obtained provide useful information for the targetedcancer therapy.Methods:(1) To clarify if the N-terminal ubiquitin binding domains arerequired for the function display of Usp28, the enzymatic catalysis experiments ofthe hydrolysis of ubiquitin chains were applied to determine the enzymatic activitiesof Usp28(1-651)(containing ubiquitin/SUMO binding domains-UBA andUIM/SIM, and the catalytic domain–USP) and Usp28(121-651)(only containingthe catalytic domain-USP);(2) The canonical Ub-AMC hydrolysis assay wassubjected to determine the enzymatic activities of Usp28(1-651) and its mutants withor without the presence of SUMO1/SUMO2, and the results obtained demonstratedthat the enzymatic activity of Usp28is cross-regulated by the non-covalent bindingof SUMO1/SUMO2.(3) The solution structure of Usp28(1-120) solved by usingmultidimensional heteronuclear NMR techniques, and the NMR [1H,15N] HSQCtitration data obtained by using Usp28(1-120):ubiquitin, Usp28(1-120): K48-linkeddiubiquitin, Usp28(1-120):SUMO1and Usp28(1-120):SUMO2protein sampleswere used to elucidate the underlying molecular mechanisms for the substraterecognition and the function regulation of Usp28;(4) The gene encoded the catalytic domain of Usp28was amplified by PCR (polymerase chain reaction) from humangenome, and then was subcloned into the pET vectors for protein preparationpurpose.Results:(1) With the deletion of the N-terminal ubiquitin binding domains, theenzymatic deubiquitination activity of Usp28is significantly decreased;(2)SUMO1/SUMO2down-regulates the enzymatic activity of Usp28by noncovalentlybinding to its N-terminal UIM/SIM domain;(3) The UBA domain and the UIMdomain of Usp28play key roles in the ubiquitin/diubiquitin recognition, and both ofthem bind to the β hydrophobic patch of the ubiquitin/diubiquitin;(4) The SIMdomain (SUMO interacting motif) of Usp28plays a key role in the SUMO1/SUMO2recognition, and it binds to the hydrophobic patch of the SUMO1/SUMO2;(5) Thenoncovalent binding of SUMO molecule damages the substrate recognition ability ofUsp28by shielding the interactions between the enzyme and the ubiquitin chains;(6)The DNA plasmids encoded the catalytic domain of Usp28are successfullyconstructed.Conclusion: The ubiquitin recognition ability of the N-terminal ubiquitinbinding domains (UBDs，ubiquitin-binding domains) is required for the full displayof the enzymatic deubiquitination activity of Usp28. Besides, the function of Usp28is cross-regulated by small ubiquitin-like modifier molecules (SUMOs) andubiquitin. The noncovalent binding of SUMO molecule damages the substraterecognition ability of Usp28by shielding the interactions between Usp28and theubiquitin chains, which then decreases the enzymatic activity of the enzyme.