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    Studies toward evaluating Gcn2 as a drug target : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand. EMBARGOED until 24 March 2027.
    (Massey University, 2020) Prescott, Hayley
    All living organisms are subject to dynamic environmental conditions. In order to survive a changing environment, organisms have evolved the capacity to adapt to stressors; this adaptation response occurs on both physiological and cellular levels. The majority of organisms face periods of inconsistent nutrient supply, which require cells to regulate consumption and conservation of vital resources such as amino acids and glucose. Amino acids are a fundamental nutrient essential for macromolecule biosynthesis; they are the monomeric unit of proteins, required for practically every cellular function. The amino acid stress response pathway regulates amino acid conservation when intracellular amino acid depletion occurs under physiological and pathological conditions; a key constituent of this pathway is Gcn2. Gcn2 is a ubiquitous eukaryotic protein kinase. Gcn2 is necessary for the maintenance of cellular homeostasis in response to amino acid deprivation and other physiological stressors such as UV irradiation and glucose, purine, or carbohydrate deprivation; Gcn2 not only aids survival in response to these regular physiological events but, on the other hand, is crucial for the progression of some pathologies, which include cancer. Gcn2 activity contributes to the growth and maintenance of tumours; it is, therefore, recognised as a prospective anti-cancer drug target. Inhibition of Gcn2 may be an effective strategy for impeding cancer cell proliferation and tumour growth as a stand-alone therapy or as a component of a combined therapeutic approach. In this study, we combined computational and experimental analytical methods to identify Gcn2 inhibitors and examined the effects of Gcn2 inhibition on cancer cells in combination with currently used cancer therapies.--Shortened abstract
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    Identification of large ribosomal proteins required for the full activation of the protein kinase Gcn2 in Saccharomyces cerevisiae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand. EMBARGOED indefinitely.
    (Massey University, 2019) Anderson, Reuben Andrew
    Protein synthesis is a fundamental biological process that all organisms require for maintaining life, growth and development. The maintenance of amino acid levels, the building blocks of proteins, is essential for maintaining protein synthesis under all biological conditions. Hence, amino acid shortage can be deleterious to the cell. Therefore, cells harbour mechanisms to cope and overcome amino acid starvation. When eukaryotes are subjected to amino acid starvation, the resulting accumulation of uncharged tRNAs activates the protein kinase Gcn2, leading to phosphorylation of eIF2α and activation of the amino acid starvation response. Uncharged tRNAs are the signal of starvation, directly detected by Gcn2. Gcn2 must bind to the effector protein Gcn1 and both must contact ribosomes for Gcn2 activation. The current working model for how the starvation signal is delivered to Gcn2 postulates that these uncharged tRNAs bind in the A-site of the ribosome in a codon specific manner, which are subsequently transferred to Gcn2. Gcn1 is directly involved in this process but its exact involvement is unknown. To test the working model, it is paramount to investigate where Gcn1 and Gcn2 bind on the ribosome. Ribosomes consist of a large and small subunit, each containing multiple ribosomal proteins placed in unique locations. Identification of ribosomal proteins contacting Gcn1 or Gcn2 will allow for deduction of where Gcn1 and Gcn2 bind on the ribosome. This study aimed to determine Gcn1 and Gcn2 contact points on the large ribosomal subunit, usinga genetic approach. The hypothesis was that if an interaction of Gcn1 or Gcn2 with a particular large ribosomal protein (Rpl) is important for Gcn2 activation, then its overexpression would impair Gcn2 function. Overexpression of several large ribosomal proteins impaired cell growth on a medium triggering amino acid starvation, suggesting Gcn2 activation was impaired. Groups of two or more of these Rpls were found in several regions which contain ribosomal proteins shown or suggested to interact with Gcn1 or Gcn2 previously. This included a region containing the P-stalk proteins (part of the large ribosomal complex) known to contact Gcn2. A region close to the small ribosomal protein Rps10, known to contact Gcn1, was also identified. Another region with Rpls which contacts a protein eEF3, which is suggested to share similar ribosomal contacts as Gcn1, was identified.