Eukaryotic genomes are highly organized and packaged into chromatin, a complex structure formed of proteins and DNA, in which the basic repeating unit is the nucleosome. Chromatin can be arranged in condensed or relaxed structures influencing accessibility of proteins that regulate transcription, replication, recombination and repair. One class of transiently chromatin-associated proteins is the High-Mobility Group (HMG) protein family. HMG proteins are subdivided into three subgroups: HMGA, HMGB and HMGN. HMGN1, the subject of this study, is a prominent member of the HMGN (High-Mobility Group Nucleosome-binding) protein family, the only HMG proteins that specifically binds to the nucleosomes. HMGNs are maintained in dynamic balance between nucleosome-associated and nucleosome-free pools. Regulation of chromatin involves several enzymatic activities that modify specific residues on chromatin proteins, which may influence these interactions. While associated with nucleosomes, HMGNs can interfere with some modifications of histone tails. Modification of HMGN1 on specific residues and post-translational modification (PTM) of histones are concomitantly regulated by the complex signalling networks associated with the induction of immediate early (IE) genes. Induction of IE genes is associated with phosphorylation of HMGN1 which has been suggested to increase the rate of dissociation of HMGN1 from the nucleosome, thus allowing access and modification of histone tails. My research has been focused on characterizing HMGN1 isoforms present in different cellular compartments and at different time-points during IE gene induction with various stimuli, including epidermal growth factor (EGF), anisomycin (An) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, I investigated the localization of HMGN1 within the nucleus and at specific IE gene loci, especially at sites where post-translationally modified histones are localised. In my analysis only the phosphorylation at serine 6 of HMGN1 shows a correlation with gene induction. Analysis of DNA sequences from chromatin immunoprecipitation (ChIP) has shown that HMGN1 is present at equal levels in active and inactive genes. It appears that HMGN1 localization on DNA is not dictated by a particular preference for any gene elements such as promoters, exons, introns or gene termination sequences.

The PI3K family is central to numerous cellular processes in both health and disease. The class IA isoforms of PI3K control such outputs as proliferation, metabolism and survival through their well-characterised function as lipid kinases, with their signalling thought to predominantly mediated by the Akt/PKB protein kinase. However there exist other signalling routes, including from the lipid kinase activity through other effectors, but also through a protein kinase function of the class IA isoforms themselves. Mass spectrometry is a powerful tool which has been central to the recent advances in phosphoproteomic techniques. It is now possible to use mass-spectrometry to probe the phosphoproteome of any number of systems in an unbiased and global manner. In this project, we aimed to advance our understanding of two aspects of class IA PI3K signalling which are relatively poorly understood. We used phosphoproteomic techniques which allowed us to provide answers to some old questions which have up to now proved elusive. First, we investigated the protein kinase activity of p110α. We used an in vitro protein kinase assay and coupled this to mass spectrometry techniques to identify direct substrates of p110α. We proposed two novel protein substrates and attempted to characterise them further, although we were hampered by lack of available biochemical tools. Second, we investigated the differential phosphoproteomes of the ubiquitously expressed class IA PI3K isoforms p110α and p110β in a panel of breast cancer cell lines. We used mass spectrometry-based phosphoproteomics and found significant differences in signalling between p110α and p110β in 4T1 cells, including differential regulation of previously described PI3K effectors, amongst them the Akt substrate PRAS40, and potential novel targets. Additionally, we found that some of these effects were conserved between cell lines.

Bone Morphogenetic Protein (BMP) signalling is an evolutionary conserved pathway, which functions to regulate numerous developmental processes such as cell fate determination and cellular proliferation. In Drosophila melanogaster, the ortholog of the vertebrate BMP2/4 is Decapentaplegic (Dpp). The most extensively characterised role of Dpp signalling in Drosophila is embryonic Dorsal-Ventral patterning. In this developmental environment, the Dpp morphogen acts as a step gradient to specify different concentration thresholds for target gene activation. The resulting nested domains of target gene expression in the embryo cooperate to induce the formation and subsequent maintenance of a simple extra-embryonic tissue, the amnioserosa. The amnioserosa tissue acts as an ideal model tissue to study Dpp-regulated differentiation. This study aims to identify and validate new targets of Dpp signalling, which are required for determining cell fate and differentiation of the amnioserosa tissue during embryogenesis. Additionally, this study aims to identify new regulators of the core signalling pathway. The work presented here was performed using a two tiered approach to understand in more detail the processes that regulate BMP-responsive transcription and the downstream effects. Firstly, RNA-Sequencing was performed on embryos with ectopic Dpp signalling in the early embryo. BMP-responsive target genes were idenitifed as differentially expressed when compared to control embryos. Expression studies have validated novel Dpp target genes and the list of genes that are regulated by BMP signalling has now been expanded. It can be invoked that these genes are involved in specification and/or mainentance of the amnioserosa tissue. Furthermore, I have uncovered a putative multi-tiered mechanism that exists between the Dpp and EGF signalling pathways to thus ensure correct cell fate specification and fine tuning of the Dpp signal in the Drosophila embryo. To further investigate how BMP signalling mediates such transcriptional regulation, a genome-wide RNAi screen was designed and performed to identify novel regulators of BMP transcription. Analysis of the screen data has identified a putative link between BMP-regulated transcription and transcriptional effectors of the Hippo signalling pathway, Scalloped and Yorkie. The data presented here suggests a co-regulatory requirement of these transcription factors to mediate Smad-dependent transcription.

The PTPN22 gene encoding the Lyp/Pep protein tyrosine phosphatase has recently been described as a negative regulator of T-cell receptor signalling. Little biological information is available on this protein, but a variant allele in this gene conferring a R620W change has been associated with rheumatoid arthritis and other autoimmune disease states. To gain further understanding into the roles of Lyp, this work is aimed at identifying and characterizing Lyp interactions, and elucidating the effect of the variant Lyp in immunological disease. Specifically, the interaction of Lyp with the ubiquitin ligase Cbl was further examined and characterized to uncover its role in T-cells. Furthermore, the biochemical and functional differences of the variant Lyp were examined by utilizing a murine model of the variant, Pep R619W. This work led to novel findings on the stability of the protein and its resulting dysfunction, leading to cell hyperresponsiveness. Finally, a new role for Lyp in controlling cell migration was uncovered through its interaction with GRK2. The inhibitory properties of Lyp on cell migration are disrupted in the presence of the Lyp R620W variant, leading to dysregulation of GRK2 function and altered migratory properties of cells, particularly in the collagen-antibody induced arthritis model. Understanding the normal function of Lyp, as well as dysfunction of the variant, will provide new insights into normal T-cell signalling and aid in the understanding of the processes of autoimmunity.

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule.

No description available.

The aim of this study was to investigate the role of Ca²⁺ signalling pathways in the growth, morphogenesis and hyphal reorientation responses of <i>C. albicans</i>. The genes <i>CCH1</i> and <i>MID1</i> were identified in <i>S. cerevisiae</i> as encoding putative Ca²⁺ channels. These genes have since been shown to compose a Ca²⁺ channel complex. Homologues of these genes were identified and cloned from <i>C. albicans</i>. <i>CaMID1</i> was disrupted by the Ura-blaster method, and the resulting mutant characterised. The <i>C. albicans</i> <i>mid1</i> mutant strain was sensitive to the depletion of Ca²⁺ the presence of cell wall perturbing compounds such as SDS and Calcofluor. It formed hyphae more rapidly in the presence of serum, and had a propensity to grow as elongated cells or pseudohyphae in Ca²⁺-depleted medium, on SD, or on medium containing cell wall perturbing compounds. This suggests that depletion of Ca²⁺-uptake perturbs yeast-hypha morphogenesis, perhaps by inducing a nutrient starvation stress response. The <i>mid1</i> mutant and a number of other <i>C. albicans</i> Ca²⁺ signalling mutants were defective in chlamydospore formation, suggesting a role for Ca²⁺ in two morphogenetic genesises: the hyphae and of chlamydospores. The role of Mid1p in the thigmotropic reorientation responses of <i>C. albicans</i> hyphae was investigated. The <i>mid1</i> mutant strain displayed reduced ability to reorientate growth upon contact with ridges on an etched quartz slide. Suggesting that Mid1p may function as stretch-activated Ca²⁺ channel in <i>C. albicans</i>. The ability of <i>C. albicans</i> <i>mid1</i> mutants to respond to an electric field was also attenuated, suggesting that Mid1p may form part of a voltage-sensitive Ca²⁺ channel in <i>C. albicans</i> that plays a central role in the steering mechanism of <i>C. albicans</i> hyphae. The <i>C. albicans </i>kinase Cst20p may function downstream of Mid1p in growth reorientation responses.

The PTPN22 gene encoding the Lyp/Pep protein tyrosine phosphatase has recently been described as a negative regulator of T-cell receptor signalling. Little biological information is available on this protein, but a variant allele in this gene conferring a R620W change has been associated with rheumatoid arthritis and other autoimmune disease states. To gain further understanding into the roles of Lyp, this work is aimed at identifying and characterizing Lyp interactions, and elucidating the effect of the variant Lyp in immunological disease. Specifically, the interaction of Lyp with the ubiquitin ligase Cbl was further examined and characterized to uncover its role in T-cells. Furthermore, the biochemical and functional differences of the variant Lyp were examined by utilizing a murine model of the variant, Pep R619W. This work led to novel findings on the stability of the protein and its resulting dysfunction, leading to cell hyperresponsiveness. Finally, a new role for Lyp in controlling cell migration was uncovered through its interaction with GRK2. The inhibitory properties of Lyp on cell migration are disrupted in the presence of the Lyp R620W variant, leading to dysregulation of GRK2 function and altered migratory properties of cells, particularly in the collagen-antibody induced arthritis model. Understanding the normal function of Lyp, as well as dysfunction of the variant, will provide new insights into normal T-cell signalling and aid in the understanding of the processes of autoimmunity.

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule.

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.