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Marek's Disease Virus (MDV) induces a wide range of neurological syndromes in susceptible hosts; however, the mechanisms behind the MDV-induced neuropathology are still poorly understood. The immediate-early 14kDa phosphoprotein, pp14, is associated with the neurovirulence phenotype of the virus. Yeast-two-hybrid screening identified the ER-bound transcription regulator, human CREB3 (cAMP Response Element-Binding protein), as an interacting partner of pp14, and fluorescence colocalisation between pp14 and chicken CREB3 (chCREB3) in MDV infected cells suggested an interaction between these proteins. The primary focus of this DPhil project was to further investigate this putative interaction using in vitro studies, with a view to determining if the interaction is linked to the neurovirulence of MDV. This investigation, which employed a combination of biochemical, cellular, and functional assays, found no conclusive evidence in support of the predicted interaction. In addition, this project aimed to gain structural and functional insights into the MDV neurovirulence factor pp14 and the host transcription factor, chCREB3. Biophysical characterisation of recombinant pp14B identifies pp14 as a molten globule. The results reveal the protein, while possessing substantial secondary structure, is largely disordered lacking a stable tertiary structure. Multiple lines of evidence from this study also indicate pp14 is a putative zinc-binding protein. Moreover, phosphorylation analysis of recombinant pp14B, extracted from DF1 cells, by mass spectrometry provides conclusive evidence for the presence of two phosphorylation sites in the shared C-terminal region of pp14 - serines 72 and 76 of pp14B. Structural flexibility, through a lack of a definite ordered tertiary structure, and functional features that can induce structural modifications indicate pp14 might interact with a number of binding partners and therefore could play multiple roles during MDV infection - a strong possibility due to the expression of the protein in all the different stages of virus infection. Furthermore, this thesis presents the crystal structure of the homodimeric chCREB3 bZIP. The chCREB3 bZIP possesses a structured DNA binding region even in the absence of DNA, a feature that could potentially enhance both the DNA-binding specificity and affinity of chCREB3. Significantly, chCREB3 has a covalent intermolecular disulphide bond in the hydrophobic core of the bZIP, which may play a role in promoting stability. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily possess this cysteine residue, indicating it could act as a redoxsensor. These results indicate members of the CREB3 subfamily, by possessing a putative redox-sensitive cysteine with the capacity to form an intermolecular disulphide bond, may be activated in response to oxidative stress.

Carboxylesterases are glycosylated general detoxification enzymes belonging to the serine esterase superfamily and play a critical role in the hydrolysis of numerous ester- and amide- containing molecules, including active metabolites, drugs and prodrugs. Three functionally active carboxyleterases have been identified in man (CES1-3), which all show differential tissue expression and critically overlapping, yet specific substrate selectivities. Elucidating the basis of their exact substrate preference would help facilitate the design of clinical prodrugs which are activated by carboxylesterases. Because of their widespread applications, carboxylesterases have attracted much attention in recent years, with CES1 being the most extensively studied human carboxylesterase to date. The work presented here addresses the structure-function relationship of the three human carboxylesterases using a combination of X-ray crystallography, kinetic analysis and biophysical techniques. Recombinant proteins were successfully produced using a mammalian expression system in high yield (5.0 – 84.0 mg/ L cell culture). Analytic ultracentrifugation and size-exclusion chromatography coupled to multi-angle laser light scattering were used to investigate the proteins in solution. These results showed CES1 exists primarily in a trimeric arrangement, whilst CES2 and CES3 are monomeric. Interestingly, atypical mechanisms of substrate inhibition, positive cooperativity and biphasic kinetics were observed for both CES1 and CES2. Three structures of CES1 were solved: wild type, an aglycosylated form and a catalytically inactive form, to 1.48, 1.86 and 2.01 Å respectively. The novel structure of CES2 was solved to 2.04 Å, which revealed that the enzyme forms a strand exchange dimer in contrast to the trimeric CES1. To summarise, this thesis documents a platform that has been generated for the production, characterisation and crystallization of human carboxylesterases. This will aid future structural work for protein ligand binding studies.