The prevailing view of signal transduction is that it proceeds through the linear relay of information via sequential bimolecular interactions, involving, for example, Src homology (SH) 2 domains. It has been assumed that such interactions are highly selective, i.e. that the affinities of these interactions are several orders of magnitude higher than that for non-specific interactions. However, recent studies have suggested that the difference in affinities between so-called specific and non-specific interactions is not sufficient to support such a proposal. This therefore raises the question of how signalling pathway specificity is generated at all. To address this, we have taken a systems approach by expressing and purifying >90% of the SH2 domains identified in a T cell line using a next-generation sequencing-based transcriptomic analysis, and performed a systematic survey of the interaction of these SH2 domains with a set of potential phosphorylated peptides derived from the key signalling receptors of the T cell (including CD28, CTLA-4, PD-1, ICOS, BTLA, LAT and the CD3 subunits of the TCR complex), using surface plasmon resonance-based binding assays. Our results show that, instead of being highly selective for certain SH2 domains, the T cell-expressed receptors are very cross-reactive, such that each receptor is found to interact with ~50 different SH2 domains on average. In silico analysis based on these results confirms the expectation that affinity itself is not the sole determining factor for receptor specificity. Further exploration of the system using in silico simulations incorporating the absolute concentrations of SH2 domain-containing proteins measured in T cells using a proteomics-based approach, suggests instead that the specificity of SH2 domain recruitment by T-cell receptors is the result of systems effects, with expression levels of the signalling proteins being a major factor. Surprisingly, LCK, the most highly expressed SH2 domain in resting Jurkat, is predicted to dominate the binding of most receptors, suggesting a novel mechanism of Src kinase activation and function.

DNA is a highly unstable molecule. Endogenous souces of DNA damage, such as reactive oxygen species (ROS), can cause DNA damage and it has been estimated that 20000 lesions occur in a cell per day. BER is the major pathway for the repair of these lesions and therefore maintains genome stability, thus preventing the development of human diseases such as neurodegenerative diseases and cancer. Therefore, if BER cannot accomplish the repair, accumulation of DNA damage occurs, triggering different cellular responses, such as cell cycle delay and senescence. The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating upregulation of p53 protein. Moreover, ARF expression is upregulated in senescent cells, suggesting that ARF induction might be triggerred in response to persistent DNA damage. Although ARF has been reported to be important in the regulation of proteins involved in the DNA damage response, its role is still controversial. Here, it has been shown that ARF gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage generated by disabling BER. These data suggest that PARP1-dependent poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD⁺, thus inhibiting SIRT1 activity and consequently activating E2F1-dependent ARF transcription. These findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

The phosphoinositide species phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes involved in cell growth, survival, and metabolism. The class I PI3Ks are responsible for production of PIP3, and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110, p110, p110) and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Growth of all cell types is dependent on PI3K signalling, and development of immune cells relies on a precise balance of PIP3 production. Activating mutations in the genes encoding the catalytic and regulatory subunits of PI3K lead to cancer and immunodeficiencies. The PIK3CA gene encoding the p110 catalytic subunit of class IA PI3K is one of the most frequently mutated genes in cancer, and mutations in the PIK3CD gene encoding the p110 catalytic subunit lead to primary immunodeficiency. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits (PIK3R1, PIK3R2, PIK3R3) have been identified in both cancer and primary immunodeficiencies. By asking how these mutations mediate activation and disease phenotypes, we can identify the natural regulatory molecular mechanisms of class IA PI3Ks. Fundamentally understanding how mutations in PI3K subunits mediate human disease will expand our knowledge of PI3K biology and is essential to the development of novel therapeutics. To identify the molecular mechanisms of class IA PI3K activating mutations, I employed a sophisticated combination of hydrogen-deuterium eXchange mass spectrometry (HDX-MS) with biochemical activity assays to probe the regulatory mechanisms of PI3Ks. HDX-MS measures the exchange rate of amide hydrogens in solution, which in turn can provide information on protein conformation and conformational changes between different states. By comparing PI3K mutants identified in primary immunodeficiency and cancer patients to wild-type enzymes, I have identified dynamic conformational changes induced by activating mutations. Biochemical and biophysical analysis of these mutants led us to generate a panel of engineered mutations to further characterise molecular mechanisms by which class IA PI3Ks are regulated. This thesis will consist of an introduction to class IA PI3K signalling and an introduction to the method of HDX-MS, followed by two data chapters wherein I investigate the mechanisms of activating mutations in PIK3CD followed by an investigation into activating mutations in PIK3R1. A conclusion and discussion of future directions will be presented in the final chapter. This work provides novel insight into the complex regulatory mechanisms of the class IA PI3Ks, which may lead to better understanding of human diseases that activate these enzymes. / Graduate / 2020-04-06

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

Tumour necrosis factor receptor (TNFR) family members are widely expressed in cells of the immune system and are essential for the development and function of many immune cell types. The TNFR associated factor (TRAF) family are signal adapter molecules that are recruited to various members of the TNFR family and are important for the transduction of signals downstream of these receptors. In these studies, gene targeting was used to create a mouse capable of undergoing conditional inactivation of the Traf3 gene. These mice were studied alongside previously generated mice that were similarly genetically modified with respect to the Traf2 gene. The mice produced lacked expression of either TRAF2 or TRAF3 in either B or T cells. In resting B cells TRAF2 and TRAF3 were shown to cooperate to negatively regulate the signalling of B cell activating factor of the TNF family (BAFF) and its receptor (BAFF-R), the TNF ligand and receptor pair that provide obligate survival signals to B cells. Thus, TRAF2- and TRAF3-deficient B cells displayed hyperactive NF-kB2 signalling, an increased ability to survive, and almost identical gene expression profiles, emphasizing the cooperative nature of their roles in resting B cells. Importantly, the survival of these B cells was completely independent of BAFF. In normal B cells, BAFF signalling was shown to lift the negative regulation of survival mediated by TRAF2 and TRAF3, by depleting TRAF3 from the cell. This process was shown to require TRAF2. T cells deficient in TRAF2 or TRAF3 also displayed hyperactivity of the NF-kB2 pathway, but they did not accumulate in vivo or show extended survival in vitro. Mice lacking TRAF2 or TRAF3 in their T cells did however display a decrease in the number of memory phenotype CD8+ T cells. These studies indicate that some of the roles of TRAF2 and TRAF3 are common between B and T cells. However, the consequences of loss of TRAF2 or TRAF3 in B and T cells differs considerably, presumably due to the differential TNFR expression and usage by each cell type.

Patched-1 (Ptch1) is the primary receptor for Hedgehog (Hh) ligands and mediates both canonical and non-canonical Hh signalling. Previously, our lab identified that mice possessing a Ptch1 C-terminal truncation display blocked mammary gland development at puberty that is overcome by overexpression of activated c-src. Testing the hypothesis that this involves a direct interaction between Ptch1 and c-src, we identified through co-immunoprecipitation that Ptch1 and c-src associate in an Hh-dependent manner, and that the Ptch1 C-terminus regulates activation of c-src in response to Hh ligand. Since the effects of Ptch1 intracellular domain deletions on canonical Hh signalling are ill-defined, we assayed this through luciferase reporter assays and qRT-PCR. Transient assays revealed that the Ptch1 middle intracellular loop is required for response to ligand, while qRT-PCR from primary cells showed that C-terminal truncation impairs canonical Ptch1 function. Together, this indicates that the intracellular domains of Ptch1 mediate distinct canonical and non-canonical functions.

Patched-1 (Ptch1) is the primary receptor for Hedgehog (Hh) ligands and mediates both canonical and non-canonical Hh signalling. Previously, our lab identified that mice possessing a Ptch1 C-terminal truncation display blocked mammary gland development at puberty that is overcome by overexpression of activated c-src. Testing the hypothesis that this involves a direct interaction between Ptch1 and c-src, we identified through co-immunoprecipitation that Ptch1 and c-src associate in an Hh-dependent manner, and that the Ptch1 C-terminus regulates activation of c-src in response to Hh ligand. Since the effects of Ptch1 intracellular domain deletions on canonical Hh signalling are ill-defined, we assayed this through luciferase reporter assays and qRT-PCR. Transient assays revealed that the Ptch1 middle intracellular loop is required for response to ligand, while qRT-PCR from primary cells showed that C-terminal truncation impairs canonical Ptch1 function. Together, this indicates that the intracellular domains of Ptch1 mediate distinct canonical and non-canonical functions.

The NF kappa B family of heterodimeric transcription factors is activated by many stimuli, and lead to the upregulation of countless genes. Not surprisingly, NF kappa B plays a critical role in many aspects of cellular function. In T and B lymphocytes, antigen receptor stimulation leads to the activation of NF kappa B through a signal transduction cascade involving the Bcl10-MALT1 complex. We hypothesized that this complex may be critical to signalling cascades other than those emanating from antigen receptors. B cell activation factor of the TNF family (BAFF) activates non-canonical NF kappa B heterodimers that promote B cell survival. Here, we show that MALT1 is required for BAFF-induced phosphorylation of NF kappa B2 (p100), p100 degradation and RelB nuclear translocation in B220+ B cells. TRAF3, a known negative regulator of BAFF-R mediated signaling, interacts with MALT1 in a manner which is negatively regulated by BAFF, and TRAF3 levels are enhanced in MALT1-/- B cells. MALT1-/- CD21highCD23low (MZ) B cells show a defect in BAFF-induced survival and MALT1-/- x BAFF-transgenic (Tg) mice have decreased MZ and B1 B cell levels compared to BAFF-Tg mice. In agreement with this in vitro data, phenotypes associated with over-expression of BAFF including increased serum immunoglobulin titres, spontaneous germinal center (GC) formation, and immune complex deposition in the kidney were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction. The mechanism by which the Bcl10-MALT1 complex regulates antigen induced NF kappa B activation in T cells remains controversial. To shed light on this regulatory network, we conducted biochemical purification of Bcl10, and identified Uev1a, a known regulator of antigen receptor mediated NF kappa B activation. We hypothesized that mms2, and structurally similar molecule to Uev1a, may also impinge on NF kappa B activation. Mms2 overexpression in 293T cells inhibited the Bcl10-induced activation of an NF kappa B sensitive luciferase. Lymphocyte development and antigen receptor induced activation occurs normally mms2-/- mice. However, class switched serum immunoglobulins, and survival responses to DNA damage inducing gamma-irradiation, are decreased in mms2-/- mice. Therefore, mms2 is dispensible in vivo for lymphocyte function and development, but is required for DNA damage responses.

Tumour necrosis factor receptor (TNFR) family members are widely expressed in cells of the immune system and are essential for the development and function of many immune cell types. The TNFR associated factor (TRAF) family are signal adapter molecules that are recruited to various members of the TNFR family and are important for the transduction of signals downstream of these receptors. In these studies, gene targeting was used to create a mouse capable of undergoing conditional inactivation of the Traf3 gene. These mice were studied alongside previously generated mice that were similarly genetically modified with respect to the Traf2 gene. The mice produced lacked expression of either TRAF2 or TRAF3 in either B or T cells. In resting B cells TRAF2 and TRAF3 were shown to cooperate to negatively regulate the signalling of B cell activating factor of the TNF family (BAFF) and its receptor (BAFF-R), the TNF ligand and receptor pair that provide obligate survival signals to B cells. Thus, TRAF2- and TRAF3-deficient B cells displayed hyperactive NF-kB2 signalling, an increased ability to survive, and almost identical gene expression profiles, emphasizing the cooperative nature of their roles in resting B cells. Importantly, the survival of these B cells was completely independent of BAFF. In normal B cells, BAFF signalling was shown to lift the negative regulation of survival mediated by TRAF2 and TRAF3, by depleting TRAF3 from the cell. This process was shown to require TRAF2. T cells deficient in TRAF2 or TRAF3 also displayed hyperactivity of the NF-kB2 pathway, but they did not accumulate in vivo or show extended survival in vitro. Mice lacking TRAF2 or TRAF3 in their T cells did however display a decrease in the number of memory phenotype CD8+ T cells. These studies indicate that some of the roles of TRAF2 and TRAF3 are common between B and T cells. However, the consequences of loss of TRAF2 or TRAF3 in B and T cells differs considerably, presumably due to the differential TNFR expression and usage by each cell type.

Our immune system must be able to rapidly fight against pathogens, but at the same time be tightly regulated to prevent harmful autoimmune and inflammatory responses. This intricate balance is controlled in part by T lymphocytes. Therapies targeting T cells have the potential to revolutionise the ways in which inflammation and autoimmune diseases are treated. However, before this can be achieved, a better quantitative understanding of the molecular processes controlling the functions of these cells is required. T cell signalling is tightly regulated by a series of complex molecular networks, which converge on key transcription factors, including Nuclear Factor-κB (NF-κB), Nuclear Factor of Activated T cells (NFAT), and Activator Protein 1 (AP-1). Using a combination of single-cell time-lapse imaging, and genome-wide assays probing for chromatin accessibility and gene expression, this study provides a better understanding of the mechanisms underpinning T cell activation and signalling. One central tenet of T cell activation is that activation-associated gene expression is triggered by the binding of the cognate antigen to the T cell receptor (TCR), and enhanced by co-stimulatory receptors, including CD28, which act to augment TCR signalling. This study shows that activation- associated gene expression programmes (induced by calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) in Jurkat T cells) are closely associated with specific chromatin landscapes. Further to this, data shown here indicate that the integration between TCR and co- stimulatory receptor signalling occurs at the chromatin level, and plays a pivotal role in regulating T cell activation. Using live-cell imaging, this study also shows that information about the diverse external signals received by T cells could be encoded within the dynamic nuclear translocations of key transcription factors. In particular, TCR signals appear to be processed by the duration of NFAT nuclear occupancy. TCR stimulation in the presence of a co-stimulatory signal resulted in the rapid nuclear import and export of NFAT proteins. In contrast, when TCR stimulation was applied without a co-stimulatory signal, prolonged nuclear occupancy of NFAT was observed. Further investigation suggested that the sustained activity of NFAT could confer a ‘signal memory’ within the TCR signalling network, thus providing a potential mechanism for preventing premature T cell turn-off during transient T cell-Antigen presenting cell interactions. This new detailed picture of T cell biology moves the field towards better therapeutic strategies for numerous diseases.