The ubiquitin-proteasome system regulates protein degradation. Although proteasomes localize in the nucleus of proliferating Saccharomyces cerevisiae, they are sequestered into cytoplasmic proteasome storage granules (PSG) in quiescence. Although important for cell cycle regulation and mediating external stressors, the content and structure of these membraneless PSGs remain unknown. Yeast deletion genetic screens identified several ubiquitin-related genes involved in proteasome sequestration into PSGs. This study aims to determine whether changes in free ubiquitin levels or ubiquitin post-translational modifications affect proteasome dynamics in quiescence. Unlike the wild-type, PSGs were not seen in catalytically inactive Ubp6 mutant strains in quiescence and proteasomes failed to be imported into the nucleus upon the resumption of cell growth. Although no significant differences in proteasome configurations were observed, Western blot analysis of these mutants suggests the presence of post-translationally modified monoubiquitinated proteasomes in quiescence. Ubiquitin modification may target proteasomes towards lysozomal degradation rather than into PSGs for storage.

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.

Ability to diagnose cancer before it progresses into advanced stages is highly desirable for the best treatment outcome. A sensitive test to analyze complex samples for specific cancer biomarkers would provide with important prognostic information and help to select the best treatment regimen. A highly robust, ultra sensitive and cost-effective electronic chip platform was used to detect nucleic acid biomarkers in heterogeneous biological samples without any amplification or purification. Chronic myelogenous leukemia (CML) was chosen as a model disease due to its hallmark genetic abnormality. This disease state therefore has an ideal market to test the detection of the fusion transcripts in complex samples, such as blood. It was shown that the CML-related fusion can be detected from unpurified cell lysates and as low as 10 cells were needed for detection. Finally, patient samples were analyzed using the assay and the fusion transcripts were accurately identified in all of them.

The lysine-dependent acid stress response system in Escherichia coli protects the cells under moderately acidic conditions. It consists of LdcI, the inducible lysine decarboxylase and CadB, the lysine-cadaverine antiporter. LdcI interacts with both ppGpp, the signalling molecule in the stringent response, and RavA, a MoxR-family AAA+ protein induced in the stationary phase. Experiments in vitro have shown that ppGpp inhibits the activity of LdcI and that the interaction between LdcI and RavA antagonizes the inhibition. In this work, it was demonstrated, by using a media shift assay, that the antagonistic regulation of RavA and ppGpp of LdcI activity also takes place in vivo, thereby linking acid resistance to the stringnent response. As part of this study, components of the lysine decarboxylase pathway and the ravA-viaA operon were endogenously tagged with fluorescent proteins. These strains are useful tools to study the localization behaviour of these proteins under different stress conditions.

The Hippo pathway plays a key role in controlling organ growth and size. In mammals, core pathway components include the Lats1/2 and Mst1/2 kinases, which phosphorylate the transcriptional regulators, Taz and Yap. To identify novel upstream pathway regulators high throughput protein-protein interaction screens, called LUMIER (LUminescence-based Mammalian IntERactome) were performed together with a functional screen using a luciferase reporter that examines Hippo pathway responses. The screens revealed 1103 protein-protein interactions and 227 transcriptional regulators, which were particularly enriched in cytoskeletal regulators. A subset of these hits including BTK, Dvl1, Dvl2, Dvl3, Ing2, Magi2, Mark4 and Trip6 were validated by manual LUMIER assays and co-immunoprecipitation (Co-IP). Of particular interest was the microtubule dynamics regulatory protein MARK4. Loss of Mark4 prevents Taz activity demonstrating its role as a potential negative regulator of the Hippo pathway. Further studies could help decipher mechanisms of how Mark4 and the other cytoskeletal hits act to modulate the Hippo pathway.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.