Proteins in health and disease: A structural approach to understanding cell mechanisms

Proteins perform a wide range of cellular and extracellular functions, and these functions in many cases are governed by the protein structure. From antibodies, that help to protect the host from viruses and bacteria, transport proteins, which direct proteins to discreet locations within the cell, through to enzymes that perform thousands of chemical reactions, proteins are highly complex macro molecules that are governed by their structural architecture. Whilst proteins perform a plethora of cellular functions and are critical for cell growth and development, proteins are also able to mediate cancer progression, disease pathogenesis, and microbial infection. A clear understanding of protein structure and function is critical for understanding not only the underlying basis of health and disease processes, but also present novel targets for therapeutic intervention. This study aimed to characterise, via high resolution structural approaches, the molecular basis through which proteins mediate three distinct cellular mechanisms; cancer stem cell progression, bacterial pathogenesis, and viral infection. Macromolecular X-ray crystallography was chosen as a main tool for structural studies, and used in conjunction with several supplementary methods to understand and evaluate the mechanisms of cell function.This thesis has established the specificity for henipavirus W protein through extensive experiments using protein crystallography and mutational analysis. Understanding of W protein nuclear import as well as inhibition of nuclear import via interaction with transport adapter proteins obtained in this study will deliver in-depth information on W protein’s roles in the nucleus as well as provide strong basis for potential novel therapeutics. Evaluation and characterisation of W protein with novel biding partner 14-3-3 protein in relation to nuclear import mechanism has also been evaluated. LSD1, an enzyme responsible for demethylation of histone and other nuclear targets, plays important roles in epigenetic reprogramming that occurs when sperm and egg come together to make a zygote (Adamo et al., 2011; Morgan, Santos, Green, Dean, & Reik,2005). Deletion of the gene for KDM1A can have effects on the growth and differentiation of embryonic stem cells (Di Stefano, Ji, Moon, Herr, & Dyson, 2007; Rudolph et al., 2007). Deletion in mouse embryos is lethal; embryos do not progress beyond Day 7.5 (Shao, Ding,& Gong, 2008; Wang et al., 2007). KDM1A has also been shown to play a role in cancer progression, aggressiveness, and increased risk of cancer relapse (Lim et al., 2010; J. Wu,12Hu, Du, Kong, & Pan, 2015).. Therefore, the inhibition of KDM1A may be a possible treatment for cancer (Hosseini & Minucci, 2017). Findings from this thesis validated previous LSD1 inhibitor analogues such as GSK2879552, ORY-1001, and phenelzine sulfate that have shown to covalently modify the FAD cofactor. In addition to covalent modification of FAD, phenelzine sulfate has shown to disposition LSD1 TOWER domain hence hindering the interaction with CoREST. This thesis also evaluated the inhibition capacity and mode of action of novel in-class inhibitor of LSD1 nuclear localisation pathway - L1 and cL1 peptides. Targeting nuclear import of LSD1 via inhibition of the interaction with nuclear transport adapter proteins provide a new alley for development of first-in-class inhibitors.This dissertation contains the detailed structural and functional analysis of first described Gram-negative B. thuringiensis SpeG protein and confirms the presence of this unique enzyme in both Gram positive and Gram negative bacteria. In addition, this work evaluates the importance of the key region of SpeG protein for its ability to oligomerise and exhibit its unique allosteric mechanism when compared to other n-acetyltransferases. The role of SpeG in pathogenic bacteria is implicated in various stages of bacterial pathogenesis and survival, therefore understanding of structure-function relationship of this protein is beneficial for providing the basis for the development of novel therapeutic strategies. Overall, this thesis provides solid basis and understanding of several cell mechanisms and proteins involved during health and disease. Described specificity and inhibition mechanisms play important roles in cell physiology and more attention needs to be placed on further potential inhibition mechanisms as this can be utilised as a solid platform for novel treatment and therapeutics development.