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    Unraveling the dynamics of protein-protein interactions in the Gcn2 signal transduction pathway : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics, Massey University, Albany, New Zealand
    (Massey University, 2016) Ramesh, Rashmi
    Eukaryotic cells regulate protein synthesis (translation) for a rapid response to various types of stress, and this involves several protein-protein interactions (PPIs) and protein phosphorylation. Phosphorylation of eukaryotic initiation factor-2 α (eIF2α) is a common regulatory mechanism to adjust protein synthesis in response to various stimuli. Gcn2 (General Control Non-derepressible) is an eIF2α kinase that is conserved from yeast to mammals, that is activated in response to amino acid starvation. Gcn2 activation leads to a reduction in global protein synthesis and simultaneous augmented translation of GCN4, a transcriptional activator of genes that are necessary to overcome stress. This cascade of events that allows cells in stress adaptation constitutes the General Amino Acid control (GAAC) pathway in yeast. Gcn2 activity is controlled by a large array of proteins that directly or indirectly regulate Gcn2. Gcn2 has to bind another protein called Gcn1, in order to be activated in response to amino acid starvation. Yih1 (Yeast IMPACT homolog 1) in yeast and its counterpart IMPACT (IMPrinted and AnCienT) in mammals are homologous proteins that indirectly regulate Gcn2. Yih1/IMPACT inhibit Gcn2 by competing for Gcn1 binding. Yih1 associates with Actin, and studies so far have suggested that Yih1 only inhibits Gcn2 when it dissociates from Actin. The focus of this thesis work was to shed more light on those interactions relevant for Gcn2 regulation. Firstly, we have identified that the Yih1 mediated interactions occur at distinct cellular locations within the cell, supporting the idea that spatially restricted cellular interactions controlled Gcn2 function. Using in vitro studies we have identified the regions on eEF1A that are involved in Gcn2 and Yih1 binding. The distinct binding sites for both proteins on eEF1A led to further investigations on how the dynamics of these interactions involving eEF1A might affect Gcn2 function. Together with unpublished observations by E Sattlegger and B Castilho, a function for the Yih1 ancient domain in interacting with eEF1A has been identified. Finally, the mechanisms by which Actin might control Gcn2 function were studied. In this regard, we have identified the Yih1-Actin interaction as one of the key PPIs involved in the crosstalk between the cytoskeleton and Gcn2 regulation. Together, the findings presented in this thesis, support the hypothesis that Gcn2 activity is spatiotemporally controlled by dynamic PPIs that occur at specific time at particular locations.
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    Identification of ribosomal proteins that are necessary for fully activating the protein kinase Gcn2 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Albany, New Zealand
    (Massey University, 2014) Jochmann, Viviane Aleta
    The environment in which cells grow often changes rapidly and in order to survive, cells need to adjust their metabolic pathway to these changes. Vitally important for all organisms is the constant availability of amino acids as they are building blocks for proteins. Proteins are essential molecules involved in most biological processes in a cell. Yeast and mammals overcome amino acid limitation by switching on a signalling pathway named General Amino Acid Control (GAAC), which triggers a decrease in general protein synthesis by inhibiting translation initiation while upregulating the transcription of stress-response genes. For sensing starvation in yeast, the GAAC requires the kinase Gcn2 and its effector protein Gcn1. Gcn2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2α), which ultimately induces the selective expression of stress-response genes, leading to the de novo synthesis of all amino acids. In order to recognize the deacylated tRNA as an immediate signal for starvation, Gcn1 and Gcn2 need to be in direct contact and associated with the translating ribosome. The current model for sensing starvation by Gcn2 suggests that deacylated tRNA enters the ribosomal Asite and Gcn1 concomitantly transfers the starvation signal to Gcn2. However, the molecular details of this process are still unclear. Deletion analysis of GCN1, suggested that Gcn1 has multiple contact points with the ribosome. We therefore aim to uncover ribosomal proteins that are required to fully activate Gcn2 in order to better understand the starvation recognition process. The fact that Gcn1 has many ribosomal contact points implies that the deletion of one contact point will not remove Gcn1 from the ribosome and therefore maintains Gcn2 activation. This allows us to identify Gcn1-ribosome interaction points which are not only required to position Gcn1on the ribosome but also facilitate in Gcn1 mediated Gcn2 activation per se. Genetic studies conducted in this thesis reveal that ribosomal proteins rps18, rps26, rps28, rpl21 and rpl34 are necessary for full Gcn2 activation. The deletion of their genes resulted in an impaired growth on starvation media and in a reduction in eIF2α phosphorylation. With these results we are able to create a first map of Gcn1 contact points of the ribosome that are necessary to promote Gcn2 activation. Two ribosomal proteins that are necessary for fully activated Gcn2 are located on the large ribosomal subunit. Three others are located on the ribosomal head region of the small ribosomal subunit in proximity to the A-site region. Considering that Gcn1 is a large protein, our results support the idea that Gcn1 has multiple contact points with the ribosome and that some important contact points for Gcn2 activation are located near the ribosomal A-site.
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    Discovering links between elongation factors and general amino acid control in Saccharomyces cerevisiae : this thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy (PhD) in Biochemistry at Massey University, Auckland, New Zealand.
    (Massey University, 2011) Visweswaraiah, Jyothsna
    Continous protein synthesis is essential for life; hence, a steady supply of amino acids must be maintained. In order to respond appropriately to amino acid shortages, cells need to constantly monitor their availability. Cells have a signal transduction pathway, called the general amino acid control (GAAC), for sensing and ameliorating amino acid shortages. Since the sensing occurs on translating ribosomes, the objective of this study was to investigate links between translation elongation and the general amino acid control in S. cerevisiae. In all eukaryotes, Gcn2 and its effector Gcn1 are responsible for monitoring amino acid availability. Active protein synthesis requires eukaryotic translation elongation factors (eEFs) to associate with translating ribosomes. This study focussed on two eEFs, eEF3 and eEF1A, and their potential role in GAAC. Gcn1 has homology to eEF3, which suggests that both proteins utilise overlapping binding sites on the ribosome. Supporting this idea, it was found that over-expression of eEF3 caused sensitivity to amino acid analogues (AAAs), suppressed the growth defect associated with constitutively active Gcn2, and impaired Gcn2 function. The C-terminal domain in eEF3 was found to be responsible for affecting Gcn2 function. Over-expression of this domain was sufficient for ribosome binding and for causing AAAs. These findings suggest that eEF3 influences Gcn1 negatively. For signal perception, Gcn1 and Gcn2 need to access the ribosomal A-site where eEF1A is functional. This suggests a link exists between eEF1A and GAAC. This link was confirmed by the discovery that eEF1A interacts with Gcn2 in vivo. The Gcn2 C-terminal domain was sufficient to precipitate eEF1A, independent of ribosomes, other yeast proteins and RNA. The interaction was lost under amino acid starvation conditions, suggesting that eEF1A is a negative regulator of Gcn2 activation under replete conditions. This study reveals a link between translation elongation and GAAC. As eEF3 and eEF1A are known to interact with each other it is proposed here that they act in concert to inhibit Gcn1 and Gcn2 under replete conditions, hence suggesting a novel mechanism of Gcn2 regulation.
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    The role of the γ-glutamyl cycle in milk protein synthesis in the ruminant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Johnston, Sarah Louise
    Dairy products are New Zealand's primary export commodity. The manufacturing efficiency for dairy products would be maximised if the Dairy Industry had the ability to control milk protein production to suit the manufacture of specific products. Understanding the role of amino acid transport in regulating milk protein synthesis may allow manipulation of proteins in milk. γ-Glutamyl transpeptidase (γ-GT), an enzyme thought to play a key role in mediating amino acid transport, has been demonstrated in mammary tissue, but the role of this enzyme and its associated biochemical pathway, the γ-glutamyl cycle, has not been fully elucidated. The γ-glutamyl cycle consists of synthetic and degradative enzymes for the cysteine-containing tripeptide glutathione. γ-GT transfers the γ-glutamyl moiety from glutathione to amino acids, and has a high affinity for cyst(e)ine. The vascular supply of cysteine is thought to be insufficient to maintain milk protein synthesis. In this study, the role of the γ-glutamyl cycle in amino acid transport for milk protein synthesis was investigated using two systems, firstly, in acini isolated from the udder of lactating sheep, and secondly in lactating goats. Milk protein secretion from isolated acini significantly decreased (70%) as a result of γ-GT inhibition with acivicin, and significantly increased (250%) when supplied with cysteine as N-acetylcysteine (NAC). In lactating goats, acivicin did not affect milk yield or total protein concentration or yield, but significantly increased αs2- and κ-casein concentration in milk. This may have resulted from increased uptake of some amino acids by the mammary gland and suggests that γ-GT negatively regulates uptake of some amino acids for milk protein synthesis. NAC significantly increased milk yield, protein concentration and protein yield as a result of increased uptake of some amino acids, which may have been due to increased mammary blood flow. This increase was prevented by acivicin, however, suggesting that γ-GT plays an important role in amino acid supply. Inhibition of γ-GT may up-regulate sub-saturated transport systems leading to increased uptake of amino acids required for milk protein synthesis. Further testing of NAC and a greater understanding of the function and regulation of γ-GT may allow increased, and targeted, milk protein production as required by the Dairy Industry.
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    The role of insulin in the regulation of milk protein synthesis in pasture-fed lactating ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Back, Penelope Jane
    The primary aim of this thesis was to determine the role of insulin in milk protein production in pasture-fed lactating ruminants (ewes and cows), using the hyperinsulinaemic euglycaemic clamp (HEC) technique. Three experiments were carried out. In the first 2 experiments, the response of pasture-fed ewes and dairy cows to the HEC were established and compared to concentrate-fed ruminants (dairy cows and goats). Use of the HEC technique in pasture-fed ruminants did not result in an increase in milk protein yield or concentration. However, a reduction in feed intake along with maintenance of milk protein yield resulted in a change in efficiency of utilisation of dietary crude protein for milk protein production. This indicated that changes in blood insulin could result in changes in nutrient partitioning to maintain milk protein production. In Experiment 3, mechanisms were examined that could maintain milk protein production despite a reduction in feed intake. The arterio-venous concentration difference technique and a leucine tracer infusion were used to measure amino acid (AA) uptake and subsequent metabolism for milk protein production under conditions imposed by the HEC. This experiment demonstrated that the HEC reduced AA supply to the mammary gland and there was a decrease in the uptake of some AA. There was no increase in mammary blood flow to compensate for this. The deficit in the ratio of AA uptake to their secretion in milk protein suggests the use of plasma free AA concentrations underestimates uptake of AA by the mammary gland and there are contributions by alternative sources such as peptide AA and erythrocytes. There was no decrease in leucine oxidation in the mammary gland, indicating that AA were not conserved for milk protein production through an alteration in this mechanism. These results support the theory that the mammary gland has the ability to respond to modified precursor supply to maintain milk protein output.