Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Has files: YesSubject: search.filters.subject.Protein kinases

Search Results

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Role of N-terminal domains of p400 ATPase in the ATM interaction and DNA damage response : a thesis presented in partial fulfillment of the requirements for a the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2016) Weber, Lauren Elizabeth
    Efficient repair of damaged DNA and preservation of genomic integrity is integral in the maintenance of proper cellular function and prevention of unrestricted cell proliferation. One critical threat to the stability of the genome is the double strand break (DSB), arguably one of the most cytotoxic lesions to DNA. Interference with the DSB repair mechanism can lead to dysregulation of cellular systems and the prospective development of malignancies. Two critical proteins in DBS repair are the Ataxia Telangiectasia Mutated (ATM) kinase, a serine/threonine kinase from the Phosphatidylinositol 3-Kinase-related Kinase (PIKK) family, and p400, an ATPase chromatin remodeler. ATM is one of the first responders to DSBs and is responsible for the phosphorylation of a multitude of protein substrates including the histone variant H2AX. Beyond its phosphorylation ability, ATM has been proposed as a potential shuttle for other repair machinery, aiding in the early and efficient recruitment of proteins to the DNA damage foci. One such proposed protein is p400. The exact role of p400 in DSB repair is unknown but previous studies show that there is a decrease in repair efficiency in its absence. A prospective interaction is supported by previous studies in which p400 and p400 N-terminal derivatives co-immunoprecipitate with ATM in vivo in HEK293T cells. This study aimed to confirm the interaction of ATM and p400 N-terminal derivatives in vitro and explore the functional implications of the association in vivo in U2OS cells. It was not possible to isolate full-length p400 derivatives in vitro and thus no conclusive results were obtained. Functional assays revealed the ability of one p400 fragment, F1, to inhibit DNA repair and cell proliferation after DNA double-strand break induction with bleomycin. Ectopic expression of the other two p400 N-terminal fragments, F2 and F3, induced an inhibition of cell proliferation under standard growth conditions. Although no conclusive results were acquired, a trend emerged suggesting that N-terminal fragment F1 is able to interfere with ATM protein-protein interactions resulting in a decrease in the efficiency of the DNA damage response and repair. These results implicate F1 as a potential target for further research in both DNA repair and cancer therapy.
  • Thumbnail Image
    Item
    Molecular dynamics simulations of protein-membrane interactions focusing on PI3Kα and its oncogenic mutants : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Computational Biochemistry at Massey University, Albany, New Zealand
    (Massey University, 2017) Irvine, William A
    The interactions between proteins and membranes are key to many aspects of biological function. Molecular dynamics simulations can provide insight into both atomic-level structural details and energetics of protein-membrane interactions. This thesis describes the development of a physiologically accurate brain lipid bilayer, and its use in molecular dynamics simulations to characterise how proteins that are important drug targets interact with the cell membrane. A method for rapidly identifying the orientation of a protein that interacts most favourably with a membrane was also developed and tested. The first chapter provides an introduction to molecular dynamics and its role in the context of this research. The second chapter details the development of a cellular membrane with a physiologically representative brain lipid composition. This was done through the testing of simple systems prior to the construction of two more complex lipid bilayers comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositide 4,5 bisphosphate (PIP2), sphingomyelin, and cholesterol. The third chapter implements the brain lipid bilayer in the development of a rotational interaction energy screening method designed to predict the most favourable orientation of a protein with respect to the cellular membrane. The functionality of the method was validated through application to two membrane proteins commonly implicated in cancer: the phosphatase and tensin homolog (PTEN), and the p110α-p85α phosphatidyl-inositol kinase (PI3Kα) complex. The fourth chapter corresponds to the main focus of this research, the behaviour of wild type PI3Kα and two of its oncogenic mutants (E545K and H1047R) with regards to membrane and substrate interaction. It was primarily found that H1047R’s increased membrane affinity allowed it to sample a catalytically competent orientation independently of Ras, unlike the wild type. Furthermore, it was also found that the position of the C terminal tail with regards to the substrate binding pocket was crucial in the achievement of a catalytically competent position against the cellular membrane. The fifth and final chapter describes a cytochrome P450 system embedded in a cellular membrane. It was primarily found that the properties of its ingress and egress tunnels depended on the presence or absence of a substrate in the active site.
  • Thumbnail Image
    Item
    Characterisation of ERK distribution and activity in rat pheochromocytoma cells : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University
    (Massey University, 1998) MacCormick, Matthew Edgar
    Nerve growth factor (NGF) binds to the NGF receptor, TrkA, at the tips of nerve cell axons, sending a signal that prevents programmed cell death and causes survival, growth, and differentiation of the nerve cell. Both NGF and TrkA have been demonstrated to be retrogradely transported from axon tips to nerve cell bodies, however the mechanism of this transport, and its function, is strongly debated. Using a recently developed cell fractionation protocol in conjunction with in vitro reactions using an ATP regenerating system, our lab has isolated small vesicles containing NGF bound to activated TrkA. These vesicles may provide a vehicle for retrograde transport of the NGF signal and initiation of signal transduction in the cell body. ERK1 is a serine/threonine kinase that is activated by NGF-activated TrkA. Prolonged ERK1 activity is characteristic of cells stimulated by NGF. The purpose of the experiments in this thesis was to characterise the intracellular distribution and activity of ERK1 before and after NGF stimulation, in rat pheochromocytoma (PC12) cells, which are a good model for nerve cells. We have found that ERK1 activity is redistributed between cell compartments after NGF stimulation of PC12 cells. ERK1 activity increased in sedimentable fractions that emerged from mechanically permeabilised cells after NGF treatment and in vitro reactions with ATP. Importantly, the results from glycerol velocity gradient experiments showed that ERK1 was not associated with membranes. Instead ERK1 was found in a rapidly sedimenting particle whose sedimentation was not affected by detergent solubilisation. These results suggest that ERK1 is recruited into a protein complex, after activation, which may be an important step in signal transduction. Formation of this complex is likely to be downstream of signalling vesicles containing NGF bound TrkA.
  • Thumbnail Image
    Item
    A molecular genetic analysis of the requirement of TOR kinase signalling for plant growth : a thesis presented in partial fulfilment of the requirements for the degree Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2015) Rexin, Daniel
    Eukaryotes have developed a highly complex mechanism to incorporate signals from nutrient, energy, stress, developmental, and environmental cues to modulate their growth. To promote this growth, eukaryotes have to coordinate the expansion in cellular mass and size through macromolecular synthesis with the increase in cell number through division. This demands a complex orchestration of a plethora of cellular processes such as transcription, protein synthesis, metabolism and cell wall synthesis. The TARGET OF RAPAMYCIN (TOR) pathway was identified as a central integrator of this growth-regulating mechanism. Components of this pathway, including the TOR kinase and its interaction partners REGULATORY-ASSOCIATED PROTEIN OF TOR (RAPTOR) and LETHAL WITH SEC 13 PROTEIN 8 (LST8), are highly conserved among eukaryotes. This includes plants, for which the adaptation to changing environmental conditions is particularly important given their sessile lifestyle and highly plastic development. This work sought to further expand the knowledge of how TOR function was adapted to suit the requirements of plants. Therefore, I analysed genetic knock-out mutants of raptor in Arabidopsis thaliana, which resulted in a severe reduction of growth but did not cause an early developmental arrest as reported by previous studies. Detailed analysis of these mutants further revealed defects in the development of trichomes, gametophytes, and the polar extension of root hairs and pollen tubes. Potential causes for these defects were indicated by lower DNA content and limited ROS accumulation in raptor mutants. High similarities between raptor and lst8 mutants indicated that the formation of TOR complexes as found in other eukaryotes might not be functionally conserved in plants. Further, I adapted a CRE/lox system for the induction of mosaic deletions of RAPTOR, which indicated no tissue-specific requirement for RAPTOR functions within the root of A. thaliana, but demonstrated a role in the regulation of meristem size. To conclude, this data presents further evidence for an altered requirement of RAPTOR and LST8 function for TOR signalling in plants compared to fungi and animals. This thesis revealed novel functions of TOR in plant development, ROS homeostasis and endoreduplication. It further draws attention to the connection with other signalling pathways to regulate growth and development in plants.
  • Thumbnail Image
    Item
    ATM and p400 : characterisation of a novel interaction between a DNA repair enzyme and a chromatin remodeler : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2014) Smith, Rebecca Jane
    The ability to maintain genomic integrity prevents unrestricted cell proliferation and the progression of cancer. DNA repair pathways such as the DNA double-strand break (DSB) response are essential in maintaining this integrity. This system requires activation of the serine/threonine kinase ataxia telangiectasia mutated (ATM) through acetylation by TIP60, a histone acetyl transferase, and subsequent ATM autophosphorylation. During DNA repair, activated ATM phosphorylates the histone variant H2AX several kilobases either side of the break site. This phosphorylation acts a signal for additional repair proteins and chromatin remodeling complexes which repairs DNA. In a previous study, H2AX phosphorylation was induced through the over expression of TIP60 or the SWI3-ADA2-N-CoR-TFIIIB (SANT) domain of p400. It was hypothesised that over expressed TIP60 or SANT domain was able to sequester a putative negative regulator from the ATM-TIP60 complex and artificially induce activation. This study aimed to investigate if a single domain of TIP60 or if a single helix from the three helix SANT domain was responsible for the activation of the ATM-TIP60 complex. Here, the ability of the chromo domain and zinc domain of TIP60 individually and the combined zincHat domain of TIP60 to induce H2AX phosphorylation as well as three helix deletion mutants of the SANT domain of p400 was examined. While all constructs were able to be expressed in human cell lines, the induction of H2AX was variable and non-reproducible. ATM belongs to the phosphatidylinositol 3-kinase-related kinase family (PIKK). Members of the PIKK family show domain homology, where the domain of one protein is replaced with the homologous domain of another member and the function of the protein is not altered. As p400 has been previously shown to interact with TIP60 and also Transformation/transcription domain-associated protein (TRRAP), a member of the PIKK family, it was hypothesised that p400 could interact with ATM (which also interacts with TIP60). This study confirms this novel interaction between ATM and p400 through the use of co-immunoprecipitation and protein localisation using confocal microscopy. This study provides a platform to further investigate the involvement of an ATM-p400 complex during DNA repair.