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Proteins undergo post-translational modifications, such as ubiquitination and phosphorylation, which can alter their activity, localisation and stability, making a cell responsive to its internal and external environment. Ubiquitin ligases and kinases comprise large enzyme families which catalyse such reactions. The SCF-type E3 ubiquitin ligase sub-family utilise F-box proteins as the substrate targeting component. However, alongside promoting ubiquitination, the F-box protein Fbxo7 can also function as a scaffold and stabilise a subset of proteins, including Cdk6. Cdk6 is activated through binding the D-type cyclins, and historically, a key role has been as a cell cycle regulatory kinase that inactivates G1 checkpoint proteins. More recently, wider roles for Cdk6 have been identified, including as an inhibitor of glycolysis. Notably, Cdk6 has pro-survival activity in T acute lymphoblastic leukaemia (T-ALL) cells due to the phosphorylation and inhibition of glycolytic enzymes, including the rate-limiting gatekeeper, phosphofructokinase (PFKP). Our screens for Fbxo7-interacting partners identified a set of candidates that overlapped as cyclin D/Cdk6 substrates and included PFKP. Further study revealed that Fbxo7 promotes two post-translational modifications on PFKP, ubiquitination and phosphorylation, and specifically promotes Cdk6 activity. Analysis in T-ALL cells suggest that Fbxo7 inhibits the assembly of active PFKP complexes to ultimately inhibit glycolysis. This is confirmed in a murine model of reduced Fbxo7 expression, whose CD4+ T cells show higher levels of glycolytic flux, alongside various other metabolic defects including altered nucleotide biosynthesis and arginine metabolism. This places Fbxo7 as a negative regulator of glycolysis and unveils other diverse roles in metabolism, which may contribute to viability and activation defects observed in these Fbxo7-deficient murine T cells. Given that Fbxo7 negatively regulates glycolysis via PFKP, I also investigated how glucose regulates Fbxo7, as feedback loops in glucose signalling are commonplace in metabolic networks. I discovered Fbxo7 is a dose-dependent, glucose responsive protein in numerous cell types, which is both transcriptionally downregulated and targeted for autophagy in response to glucose starvation. Moreover, data suggest that Fbxo7 is responsive to other stresses, including oxidative stress, placing Fbxo7 as a nexus to link various cellular stress responses to metabolic reprogramming. In addition to PFKP, Fbxo7 has also been shown to recognise a protein called cereblon (CRBN), which is another E3 ubiquitin ligase. CRBN is of clinical relevance because its expression is required for the efficacy of immunomodulatory drugs (IMiDs) in multiple myeloma (MM), which primarily enable the recognition of neo-substrates by CRBN to elicit their anti-cancer effects. We sought to investigate a role for Fbxo7 in MM cells. We show that Fbxo7 promotes CRBN ubiquitination and propose that this targets CRBN for proteasomal degradation, which may have relevance for IMiD sensitivity. Together, these data identify two novel substrates for Fbxo7 ubiquitination and reveal a role for Fbxo7 in lymphocytes. We demonstrate that Fbxo7 expression is responsive to cellular stress and propose that Fbxo7 levels may fine-tune metabolism under different physiological and pathological conditions.

Advances in DNA sequencing technology have allowed detailed characterisation of cancer genomes and has highlighted the contribution of somatic structural variations to the mutational landscape of epithelial tumours. However, our understanding of the functional consequences of such genome rearrangements remains rudimentary. By surveying the METABRIC dataset, consisting of segmented array-CGH copy number data, and paired-end whole-genome DNA and RNA sequencing data from primary breast tumours, we found that the F-box protein encoded by FBXL17 is frequently rearranged in breast cancer. F-box proteins are the substrate-recognition components of Skp1-cullin 1-F-box protein (SCF) E3 ligases. As essential components of the ubiquitin proteasome system (UPS) they are responsible for directing target proteins for ubiquitination. Fbxl17 is a relatively understudied member of the FBXL family of F-box proteins and, in breast cancers, is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased its ubiquitination activity. Loss of the LRRs also affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway. To identify further interacting partners of Fbxl17, we performed a mass spectrometry analysis of purified Fbxl17 SCF E3 ubiquitin ligases. Co-immunoprecipitates were enriched for DNA damage/ DNA repair proteins suggesting a novel role for Fbxl17 in the DNA damage response (DDR). We have demonstrated that Fbxl17 is recruited to DNA damage sites rapidly upon double-stand break (DSB) induction and knockdown of Fbxl17 protein expression sensitises cells to the DNA damaging agent Camptothecin. Furthermore, Fbxl17 can ubiquitinate the tandem BRCT domain of the well-known DDR protein 53BP1, which we propose targets 53BP1 for proteasomal degradation. In conclusion, we have identified two regulatory networks of Fbxl17 which provide an insight into the role of Fbxl17 in breast cancer pathogenesis. These pathways may be amenable to therapeutic targeting in the future for the treatment of breast cancers rearranged in FBXL17.

The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.

Parkinson's disease is a progressive neurodegenerative disorder of the central nervous system, manifesting with both motor and non-motor symptoms. Autosomal recessive mutations in the FBXO7 gene have been identified to cause a rapidly progressing early-onset form of PD. Canonically, FBXO7 functions as a substrate-recruiting subunit of the SCF-type E3 ubiquitin ligase. However, it also has a variety of other atypical functions, such as cell cycle regulation, proteasome regulation, and mitophagy. The overall aim of this research was to characterise the functional role of FBXO7 in various in vitro and in vivo PD models. The models examined included FBXO7 shRNA knockdown SH-SY5Y cell lines, FBXO7 CRISPR knockout SH-SY5Y cell lines, primary patient fibroblasts with a FBXO7 mutation, and MEFs and tissues from a Fbxo7 KO mouse. My analysis of fibroblasts from a patient without FBXO7 expression revealed several interesting phenotypes. Briefly, the patient fibroblasts proliferated slower due to increased apoptosis and lower CDK6 and cyclin D1 expression, which led to fewer cells progressing through the G1 phase of the cell cycle. My experiments showed that these cells also had mitochondrial respiration defects, exhibiting lower basal respiration, ATP production, maximal respiration and spare capacity, in addition to complex I, III and IV deficiencies. Patient fibroblasts also had significantly lower levels of 12S and 16S ribosomal mRNA transcripts, which are necessary for the translation of mitochondrially encoded subunits of complexes I, III, and IV. Similar phenotypes were also observed in MEFs from a Fbxo7 KO mouse model, indicating conservation between human and mouse FBXO7 in regulating mitochondria, cell death and proliferation. In a tissue-specific KO mouse model of PD, where FBXO7 expression was ablated in the dopaminergic neurons, I analysed proteins regulated by FBXO7 which might be responsible for cell loss in the substantia nigra. I discovered that RPL23, a regulator of MDM2, was ubiquitinated by SCFFbxo7 using K48 chain linkages, promoting its degradation by the proteasome. This suggests that misregulation of the MDM2:p53 axis may underlie the cell loss observed in this conditional Fbxo7 KO mouse model. In conclusion, these results elaborate on the role of FBXO7 in mitochondrial biology, and identify a new ubiquitination substrate of FBXO7 in a mouse model of PD. It is hoped that by elucidating the potential pathogenic mechanisms of FBXO7 in rare familial forms of the disease, it will be possible to translate findings to the more prevalent sporadic forms of Parkinson's disease as well.