In quiescent yeast, the widespread reorganization of cytosolic proteins into punctate has been observed (Narayanaswamy et al. 2009). We seek to better understand and describe this reorganization, which we hypothesize to be a protein aggregation phenomenon. To test this hypothesis, we examined mutant proteins (Ade4p protein variants) in yeast with predicted non-native aggregation propensities and measured their punctate formation kinetics. Monitoring punctate formation kinetics involved the validation of an automated quantification technique using an Amnis ImageStream imaging flow cytometer. The automated punctate counts were strongly correlated with the manual punctate counts, with usual R² values of 0.99 or better, but evaluated 50-fold more cells per run. Fitness evaluations of the mutant yeast in the form of growth curves and batch competition experiments revealed the slowed growth of the Ade4-1286 strain and the functional inequality to the wild type strain of the Ade4-mtoin2034, Ade4-mtoin2105, and Ade4-2800 strains in competition experiments, especially when the mutants were forced to generate their own adenine. Subsequent structural analysis of the mutant proteins revealed destabilizing mutations for 4 of the 6 mutant proteins with 2 of the mutations classified as significantly destabilizing ([delta][delta]G >2 kcal/mol). We concluded that the reduction in protein fitness was likely due to the destabilizing effects of the mutations. Evaluation of the punctate formation kinetics revealed little difference between strains in the rate of punctate formation. Further examination revealed the wild type Ade4p and all of the mutants (with the exception of the Ade4-1286 mutant) were predicted to have similar aggregation propensities according to a secondary aggregation predicting algorithm (Zyggregator, Pawar et al. 2005). Additionally, solvent accessibility calculations estimate ~3-19% of the side chain surface area to be solvent accessible, which indicates proximity of mutations to the protein surface. However, mutating buried amino acids likely would have generated a greater disturbance (Matthews 1993, Tokuriki et al. 2007). We concluded that the mutations, although destabilizing, altered the aggregation propensity very little. Deletion of chaperone proteins (Hsp82p, Hsc82p, and Ssa1p) revealed no difference in the Ade4-GFP punctate formation kinetics, although a slight kinetic difference was detected in the chaperone (Hsp82p) knockout, Gln1-GFP strain and the wild type strain. While further workup is necessary in the chaperone knockout, Gln1-GFP work, the initial results are promising and suggest the involvement of protein folding machinery in punctate formation. / text

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)

Congenital myasthenic syndrome (CMS) and myasthenia gravis (MG) are, respectively, inherited or autoimmunological disorders caused by aberrant neuromuscular transmission, which manifests as fatiguable muscle weakness. A novel subtype of CMS, resulting from mutations in GFPT1 and characterised by a limb girdle pattern of muscle weakness, has been described. The gene encodes L glutamine:D fructose-6-phosphate amidotransferase 1 (GFAT1) – a key rate limiting enzyme in the hexosamine biosynthetic pathway, providing building blocks for glycosylation of proteins and lipids. The research focused on the molecular bases of the CMS resulting from mutations in the ubiquitously expressed gene, but with symptoms largely restricted to the neuromuscular junction (NMJ). The work has established a link between the NMJ and GFPT1 CMS by demonstrating that the AChR cell surface is decreased in GFPT1 patient muscle cells and in GFPT1-silenced cell lines. The decrease is likely to be caused by reduced steady-state levels of individual AChR α, δ and ε, but not β, subunits. To optimise treatment for myasthenic disorders, a comparative in vivo trial of therapy with pyridostigmine bromide and salbutamol sulphate, and pyridostigmine bromide alone, was conducted. Supplementation of the AChE inhibitor-based therapy with the β2-adrenergic receptor agonist had a beneficial effect. This offers promise for more effective treatments for CMS and MG affected individuals. Molecular causes of MG were also investigated. The search for novel antibody targets was conducted with the use of a designed cell-based assay for the detection of anti COLQ autoimmunoglobulins in MG patient sera. The antibodies were detected in 24 out of 418 analysed samples, but their pathogenicity has not been determined.