Harnessing the potential of proteins for sustainable development: from bioremediation to drug discovery

Abstract

Since the start of the human civilisation, incessant anthropogenic activities and rapid industrialisation have led to detrimental effects to the environment, human health and socio-economic prosperity. In 2015, the United Nations formulated a series of sustainable development goals (SDGs) with the aims to restore the balance in nature and to ensure a sustainable future for all. The goal of this thesis is to explore and harness the versatile nature of proteins to contribute to three of these SDGs. • Goal #3, which aims to ensure healthy lives for all • Goal #6, which aims to ensure availability of clean drinking water for all • Goal #14, which focusses on reducing marine pollution and conserving the marine ecosystems Specifically, the work in this thesis includes exploring the use of enzyme technology for environmental remediation as well as the studies of proteins and enzymes to understand the molecular mechanism that causes chronic neurodegenerative diseases, and the potential to target these protein-protein interactions for the development of new inhibitors. In chapter 2, the application of laccases for the treatment of dye-containing wastewater was explored. Novel laccases from two bacterial species (Sulfitobacter indolifex and Geobacillus yumthangensis) were studied. S. indolifex is a cold-adapted bacterium from the North Sea and G. yumthangensis is a thermophilic bacterium from a north-east Indian hot spring. The activity and stability of these laccases in different operational conditions such as pH and temperature were characterised and optimised. The ability of these enzymes to decolourise common industrial dyes was examined, which revealed they could efficiently degrade five of the seven tested dyes in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which is a redox mediator. Furthermore, the use of enzyme immobilisation technology to achieve greater operational stability and reusability for use in an industrial setting was also investigated. This work expands the arsenal of laccases available for bioremediation of dye-containing wastewater. The Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLCBc) is an enzyme that has found numerous applications in the food industry. Moreover, in medicinal chemistry it has emerged as a model enzyme for evaluation of inhibitors for human PC-PLC, which is an anti-cancer drug target. In chapter 3, the development and optimisation of a matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry-based assay to monitor activity and inhibition of PC-PLCBc was explored. Employing this assay, the use of one-phase and two-phase reaction systems to assess the inhibition of PC-PLCBc with different structural classes of inhibitors was compared. The assay results were complemented using an intrinsic protein fluorescence quenching spectroscopy-based binding assay. The advantage of the MALDI-TOF assay over the commonly used commercially available Amplex Red assay was also highlighted. This assay will therefore enable more efficient studies of phospholipases (e.g. for screening of optimised phospholipases for industrial applications) and facilitate the development of new phospholipase inhibitors. Protein-protein interactions play key roles in many important biological functions, and as such, they may hold the key for the treatment of diseases such as chronic illness. In chapter 4, the role of protein-protein interactions in Parkinson’s disease and the potential of targeting the interactions between the molecular chaperone heat shock protein 90 (Hsp90) and its co-chaperones for the development of new Hsp90 inhibitors were explored. In Parkinson’s disease (PD), the clinical mutation G2019S LRRK2 has shown to cause an increase in phosphorylation of the ribosomal protein S15. It was hypothesised that this may affect the interactions between S15 and its partner S18 in the ribosome. By using a protein pulldown binding assay and an isothermal titration calorimetry (ITC)-based binding assay, it was revealed that there were no changes in protein-protein affinity between S18 and the wild-type and phosphorylated S15. Further experiments are needed to fully understand the role of S15 phosphorylation in PD. In cancer, molecular chaperone such as Hsp90 has long been proposed to be an inhibition target for the development of new anticancer agents. However, the development of Hsp90 inhibitors is not trivial. Most compounds that are in clinical trial to date target the N-terminal domain, which may have undesired drawbacks. In this work, the ability of small molecule ligands to disrupt the interactions between the C-terminal domain (CTD) of Hsp90 and its co-chaperones were investigated. An amplified luminescent proximity homogeneous assay (Alpha) helped to identify two novel Hsp90 CTD inhibitors, paving the way for future development of new therapies for treatment of cancer. Overall, this thesis discusses the attempts to harness our knowledge in protein science and take advantage of recent developments in biotechnology and analytical chemistry to contribute towards the realisation of three SDGs (goals #3, #6 and #14). This work has successfully led to the identification and characterisation of two novel laccase enzymes that can be further developed for bioremediation purposes; developed an improved mass spectrometry-based assay that allows accurate characterisation of a phospholipase, an enzyme that has applications in the food industry and medicinal chemistry; elucidated the molecular mechanism that causes PD, and developed new inhibitors against Hsp90, a molecular chaperone that is a current inhibition target against cancers. These individual contributions add to the existing literature and in the long term will help the global effort to make the world more sustainable in the future.