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Title: Structure-function studies of intracellular and extracellular superoxide dismutases
Author: Chantadul, Varunya
ISNI:       0000 0004 9347 5063
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2020
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Superoxide dismutases (SODs) are the major components of oxidative stress defense mechanisms ubiquitously presented in wide variety of species. Three subtypes of SODs have been identified in human: SOD1 and SOD3 or extracellular superoxide dismutases (EcSOD) belong to Cu/Zn SOD family, and SOD2 belongs to MnSOD family. This study focuses on SOD1 and EcSOD in different aspects. While SOD1 is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS), EcSOD is not directly involved in any specific diseases. Indeed, EcSOD maintains the physiological level of oxidants in the extracellular environment by using its extracellular matrix binding domain. The mutations in SOD1 gene is one of the causes of the familial ALS (fALS). The SOD1 mutant, Ala4Val (A4V), contributes to 50 % of SOD1-related fALS cases in the U.S. making it the most common SOD1 mutation associated with fALS. This mutant has a greater propensity to monomerise and aggregate than any other mutants leading to the disruption of normal functions of neurons. Although the definitive treatment has not yet been discovered, several small molecules have been designed to mitigate the severity of this disease. Ebselen is an organoselenium compound and currently used in clinical trials for treatment of other diseases, probably due to its anti-oxidant property. With its versatile structure, a number of ebselen derivatives have been synthesised to enhance its therapeutic property. This study presents the ability of ebselen and some of its derivatives in promoting thermal stability of A4V SOD1 mutants when binding to Cys111 only using differential scanning fluorimetry (DSF). DSF was performed based on the C6S background due to the off-target binding of the compounds to Cys6. This provided a medium-throughput screening of new compounds. The aggregation of A4V in C6S background SOD1 in the presence of ligands was also investigated using size exclusion chromatography. The crystal structures of ebselen and its analogues bound to A4V SOD1 show that the selenium atom of the ligands covalently binds A4V SOD1 at Cys111 located at the dimer interface while there is no electron density of the ligands around Cys6 and the overall structure and monomer-monomer orientation are still conserved. Therefore, apart from its direct anti-oxidant effect, ebselen could be a promising candidate for rational drug design of fALS-related therapeutics. EcSOD is localised in the extracellular matrix to protect cells from external free radicals. The binding of EcSOD to extracellular matrix by its C-terminus is critical for maintaining the function of the organs, especially blood vessels while its N-terminus is proposed to be important for forming the quaternary structure. Structural study of the C- terminus when binding to heparin, a highly sulphated form of heparan sulphate, as well as N-terminus, is thus required for the development of potential therapeutics for several diseases. This study presents the development of purification methods of human tetrameric EcSOD from BL21 cells (eEcSOD4-mer) which involved a refolding process and high pH buffer. The identity of the purified protein was confirmed by the peptide finger print. The main characteristics of the protein: molecular weight, SOD activity and heparin binding ability, were compared with tetrameric EcSOD purified from mammalian cells (mEcSOD4-mer). The low-resolution bead models of eEcSOD4-mer were reconstructed using small angle X-ray scattering (SAXS) data. Crystallisation was performed in both eEcSOD4-mer and mEcSOD4-mer for structural study of N- and C-termini although no crystals was obtained from eEcSOD4-mer and the crystals of mEcSOD4-mer diffracted to low resolution. Several improvements may further be performed and the use of other techniques in structural study may also be considered to obtain the structure of the whole protein with its C-terminus binding to heparin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral