Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Gary Shaw

Abstract

The ubiquitin proteolysis pathway utilizes three enzymes, an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligating enzyme, to respectively activate, transfer and ligate ubiquitin (Ub) onto a substrate protein. The creation of a K48-linked poly-Ub chain on a substrate will target this protein to be degraded by the 26S proteosome. E2 conjugating enzymes are central proteins in this pathway and interact with the E1 and E3 enzymes to perform Ub transfer. The mechanism by which Ub molecules are interconnected remains poorly understood. The E2 enzymes HIP2 and Ubc1 have been shown to create poly-Ub chains in the absence of E3 enzymes and substrates. In this thesis, HIP2 and Ubc1 were investigated through physical and structural methods to clarify their mechanism of poly-Ub chain assembly.

The study of HIP2 and Ubc1 was aided by the formation and purification of stable HIP2-Ub and Ubc1-Ub disulphide linked complexes that closely resemble the HIP2~Ub and Ubc1~Ub thiolester intermediates. The physical techniques of sedimentation equilibrium and SAXS determined that HIP2 and Ubc1 as well as their disulphide complexes are predominantly monomeric. Activity assays were also performed on these enzymes indicating that the E2~Ub thiolester is the sole species required to create poly-Ub chains. Additionally, these assays determined that both free Ub and E2-Ub complexes could act as Ub acceptors for poly-ubiquitin chain extension. NMR experiments were also performed through the use of isotopically labelled HIP2, HIP2-Ub and HIP2-Ub2 complexes. NMR chemical shift perturbation experiments identified significant intramolecular interactions between HIP2 and Ub in both HIP2-Ub and HIP2-Ub2 complexes. The intramolecular interaction within HIP2-Ub2 utilizes a C-terminal Ub-associated (UBA) domain and this domain is not present in other human E2 enzymes. These intramolecular interactions indicate the HIP2-Ub and HIP2-Ub2 complexes behave predominantly as Ub donors within poly-Ub chain formation. These results have allowed the formulation of mechanisms to describe HIP2 and Ubc1 function. The determination of these mechanisms is especially important for HIP2, as its function has been associated with the progression of both Huntington’s and Alzheimer’s disease.

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