Biochemistry and Pharmacology - Theses
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ItemInvestigating the contribution of the cysteine protease legumain to oral squamous cell carcinoma progression( 2022)Oral cancer is the eighth most common form of cancer worldwide. The mechanism behind oral carcinogenesis is poorly understood and current treatment strategies are ineffective. Survival rates drop significantly once metastasis develops, and predicting which cancers will go on to metastasise is currently not possible. Further mechanistic studies are thus required to improve patient outcomes. Legumain is a cysteine protease that has previously been reported to contribute to the carcinogenesis of several cancers. We have recently demonstrated that legumain contributes to orofacial pain in mouse models of oral cancer, via cleavage of protease-activated receptor-2 (PAR2). We hypothesised that legumain may also contribute to other features of oral carcinogenesis. Using the legumain-selective activity-based probe LE28, we measured legumain activity in human oral squamous cell carcinomas (OSCC) and patient-matched normal oral mucosa. We found that legumain activity was strongly upregulated in tumours of all patients examined, compared to normal oral mucosa. We further validated this finding using tumour samples from two murine models of oral cancer: orthotopic xenografts of HSC-3 human OSCC cells and 4NQO carcinogen-induced OSCC. Immunohistological analysis of samples from these mice revealed that legumain is expressed in both tumour and stromal cells and is predominantly located at the invasive edge of tumours. We examined legumain levels in a panel of human OSCC cell lines, as well as in macrophages primed by conditioned media (CM) from these cells. Compared to normal oral keratinocytes, cultured human oral cancer cells express higher levels of active and total legumain, as do macrophages exposed to tumour CM. We next analysed the polarisation of CM-exposed macrophages by flow cytometry and qPCR and found that these cells were polarised towards a classical M1 activation state. Using CRISPR-Cas9 technology, we generated legumain-deficient HSC-3 and SCC-9 cells and analysed them for features of carcinogenesis in vitro and in vivo. Compared to wild-type cells, legumain knockout cells exhibited reduced proliferation. When legumain-deficient HSC-3 cells were inoculated subcutaneously in mice, the resulting tumours exhibited >90% reduction in volume and less vascularisation compared to tumours generated from wild-type cells. Thus, it appears that tumour-derived legumain promotes oral cancer growth. We next used unbiased bottom-up proteomics analysis to identify changes in protein expression between wild-type and legumain knockout HSC-3 cells. Pathways involved in energy metabolism were highly dysregulated in legumain knockout cells compared to wild-type cells. A cancer signalling phospho protein array on subcutaneous xenograft HSC-3 tumours revealed changes in metabolic pathways in legumain knockout tumours compared to wild-type tumours. Collectively, these results demonstrate that legumain is highly activated in the oral cancer microenvironment and may contribute to tumour growth and invasion. Future studies aim to distinguish the proteolytic and non-proteolytic functions of legumain in the oral cancer microenvironment as well as its cell-specific roles in oral cancer progression. Ultimately, we aim to determine whether legumain inhibitors will have therapeutic value, resulting in improved outcomes for oral cancer patients.
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ItemStructure and dynamic studies of AAA+ ATPase p97 molecular machine( 2021)The superfamily of AAA+ ATPases (ATPases Associated with diverse cellular Activities) is a conserved and vital family of proteins in all living organisms. AAA+ ATPases hydrolyse ATP and convert the chemical energy to mechanical force. These mechanochemical enzymes perform a variety of cellular functions. Members of this superfamily most commonly assemble as homohexamers, the functional form of these macromolecules. A prominent member of this family is the protein named p97, or VCP. In all eukaryotic cells, p97 is one of the most abundant cytosolic proteins where it plays a central role in organelle and protein homeostasis in ubiquitin-dependent and independent pathways. It acts as an unfoldase to unfold its substrate. To play a role as a multifunctional protein in the cell, p97 interacts with a vast number of cofactors. The monomer of the homohexameric AAA ATPase p97 contains the cofactor or substrate-binding domain (N) and two ATPase domains (D1 and D2), and a disordered C terminal region. A single amino acid substitution in this protein causes neurodegenerative diseases, and an increased expression level of p97 has been identified in several cancers. It also plays a role in viral infections. Therefore, it is an important therapeutic protein. The structure of p97 has been widely investigated, and the high-resolution X-ray and cryo-EM structural information of full-length p97 became available in recent decades. However, structural knowledge and detailed insight into the disease-associated mutant, and the interaction of p97 with cofactors have limited our understanding of how disease-associated mutation and cofactors regulate or modify the functions of p97. The work presented here attempts to characterise how one of the most clinically relevant mutations of p97 (R155P) and one of the best-characterized cofactors (p47) modify the dynamic properties of p97. My approach has been to combine a series of structural and biochemical techniques such as single-particle cryo-EM, HDX-MS, X-link MS, and 19F NMR. In chapter 2, I describe how I have determined the high-resolution cryo-EM structures of the R155P and wild-type p97 in interaction with ATPgS. In chapter 3, using a combination of the structural and dynamical approaches, I show that the mutant stabilizes the up conformation of the N domain and affects the intra and inter subunit communication of the p97. During these studies, I have captured the wild-type p97 protein in a novel conformation and identified that the conformation of the N domain is not solely determined by the nucleotide occupancy of the D1 and D2 domains. In chapter 4, I present evidence I collected using SEC-MALS, AUC, and SPR that support the fact that the cofactor p47 is a monomer in solution. This analysis addresses controversy in the field. Using HDX-MS and X-link MS, I have shown changes in the conformations and interacting domains of both p97 and p47 upon complex formation with and without adding ATPgS. In chapter 5, I present preliminary studies on how 19F NMR would be an appropriate approach to study the dynamic flexibility of the p97 N domain. Together, these studies provide additional knowledge to the molecular mechanisms that regulate p97 function, our work will lead to progress in using hybrid structural biology techniques to understand the mechanism of macromolecules’ function.