Theses and Dissertations at Montana State University (MSU)
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Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item The relationship between physiological stress response and variation in omics data(Montana State University - Bozeman, College of Letters & Science, 2021) Steward, Katherine Fay; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Omics analysis is the cornerstone of systems biology. It offers comprehensive assessments of stress, interaction networks and connections to phenotype. Defining a stressed phenotype can be challenging, however, as stress response mechanisms can arise from a range of environmental conditions and experimental perturbations. Previous work from our lab noted the possibility of a relationship between stress in omics data and the variation of that data. This connection has yet to be clearly defined, and the cellular mechanisms responsible for the canalization of omics data remain a mystery. In this work I have taken advantage of the sensitivity of metabolomics and proteomics to detect cellular stress and characterize its relationship to variation. By utilizing coefficient of variation (CV) as a statistic of merit, the depth of the relationship between stress and variation can be uncovered. Once the model was clearly defined, a proteomics dataset with a large proportion of protein coverage was utilized to investigate what pathways might be responsible for the metabolite and protein canalization.Item Proteomics analysis of the metabolic transition between aerobic and anaerobic conditions in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) Refai, Mohammed Yahya; Chairperson, Graduate Committee: Brian Bothner; Nina Paris, Hunter Fausset, Monika Tokmina Lukaszewska were co-authors of the article, 'Proteomics analysis of the transition between aerobic and anaerobic growth conditions in Escherichia coli' submitted to the journal 'Biochimica et biophysica acta' which is contained within this dissertation.As a facultative anaerobe, Escherichia coli has the ability to grow in anaerobic and aerobic environments. Despite detailed characterizations of this model organism in the presence and absence of oxygen, an in-depth understanding of changes to the proteome during transitions from aerobic to anaerobic growth is lacking. This thesis work focuses on elucidating how protein thiol oxidation and reduction change during a facultative anaerobe's transition from aerobic to anaerobic growth conditions, and pathways of thiol-mediated cell signaling. Redox driven changes in cysteine oxidation involved in signaling are referred to as 'thiol switches'. These modulate diverse biological activities ranging from gene expression and protein synthesis to environmental stress response. Surprisingly, little is known about the role of thiol switches during microbial transitions from aerobic and anaerobic growth conditions. To explore this uncharted territory, a mass-spectrometry (MS)-based proteomics workflow was developed and refined. Following extensive protocol optimization for high-throughput MS data processing, normalization, and pattern matching, the analytical pipeline was fine-tuned for the specific proteome-wide analysis of cysteine chemical modifications in E. coli. The approach was based on open-source software and publicly accessible databases, creating a transparent, reproducible, and easily sharable proteomics approach. Herein, the redox state and chemical forms of protein-based thiol switches in E. coli were characterized over time as the bacterium reversibly transitioned between aerobic and anaerobic growth conditions. Unexpectedly, differential alkylation analysis of cysteine-containing E. coli proteins revealed a higher degree of protein thiol oxidation under anaerobic growth conditions, a result not reported for E. coli or any other facultative anaerobe. Our proteome-wide analysis also revealed that cysteine redox potentials vary widely, and several specific E. coli proteins contain highly reactive thiols. These findings provide strong evidence for thiol-based signaling in E. coli in response to environmental changes such as aerobic to anaerobic growth transitions. Characterization of specific redox switches underlying metabolic changes associated with oxygen availability has uncovered a previously unknown E. coli cell signaling mechanism. Since transitioning between aerobic and anaerobic environments is associated with bacterial virulence, this work opens new avenues to target pathogenic facultative anaerobes and to develop novel thiol-based antibacterial therapies.Item Application of the thiosulfonate switch technique and a modified biotin switch technique protocols to detect protein S-nitrosothiols in mouse liver lysates in 1D and 2D gel studies(Montana State University - Bozeman, College of Letters & Science, 2015) Miller, Colin Gregory; Chairperson, Graduate Committee: Paul Grieco; Brian Bothner (co-chair)While the role of nitric oxide (NO) in cell signaling and liver growth has been well documented, the identification of S-nitrosylated proteins, one of the major NO transport mechanisms within the cell, remains a challenge. Classically, the implementation of biotin labeling, known as the biotin switch technique (BST), with streptavidin-agarose bead pulldown and subsequent immunoblotting, has offered the best results for identifying S-nitrosocysteine residues within proteins. However, this technique has come under scrutiny for its use of ascorbate as a reducing agent. Numerous published accounts have shown ascorbate's poor reducing potential especially for S-nitrosoproteins. To this end, the Grieco lab has shown that pure S-nitrosylated proteins can be transformed into S-phenylsulfonylcysteine residues, which can be readily converted into mixed disulfides thus allowing for labeling of pure nitrosylated proteins at pH's as low as pH 4. This protocol is referred to as the thiosulfonate switch technique (TST). The Grieco lab has also modified the biotin switch technique to incorporate electrophilic maleimide and orthopyridyl disulfide (OPSS) dyes for fluorescence labeling of the S-nitroso proteome. To examine the scope and limitations of the TST vis-a-vis cell lysates, the TST protocol and the modified BST protocol, both employing novel Z-CyDyes developed in the Grieco Laboratory, have been used to specifically label S-nitrosylated proteins in complex liver lysates. The successful labeling of mouse liver lysates, employing UV and ascorbate SNO knockout negative controls, is demonstrated in both 1D and 2D gel studies. Also reported herein is the creation of novel second generation maleimide dyes (SO3-Cy-Mal) based on Z-CyDyes.Item Toward resolving the human neocortex epileptic proteome(Montana State University - Bozeman, College of Letters & Science, 2013) Keren-Aviram, Gal; Chairperson, Graduate Committee: Edward DratzEpilepsy is a common and often devastating neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication can benefit from surgical removal of brain regions that produce epileptic activities. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study, as part of a Systems Biology of Epilepsy Project, which utilizes in vivo electrophysiologically-characterized human brain samples from the neocortex of 6 patients with refractory epilepsy, to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. This study is unique in that comparison of protein expression was made within same patient, between nearby epileptic and non-epileptic (or less epileptic) brain regions, as defined by their interictal (between seizure) spike frequencies. Protein spots were resolved from three subcellular fractions, using two-dimensional differential-in-gel-electrophoresis, revealing 31 spots that changed significantly and were identified by liquid-chromatography tandem mass-spectrometry. Interestingly, glial fibrillary acidic protein was found to be consistently down regulated in high spiking brain tissue and glial fibrillary acidic protein levels showed strong negative correlation with spiking frequency. We next developed a two-step analysis method to select for frequently changing spots among the patients and identified 397 of those proteins. Spots of interest were clustered by protein expression patterns across all samples. This analysis predicted proteomic changes due to both histological differences and molecular pathways by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial and vascular changes and changes in cytoskeleton and neuronal projections that provide new insights into the structural and functional basis of neocortical epilepsy.Item Synthesis of zwitterionic cyanine dyes for use in proteomics(Montana State University - Bozeman, College of Letters & Science, 2012) Epstein, Mark Galen; Chairperson, Graduate Committee: Paul GriecoThe CyDye family of fluorescent dyes are the tools currently in use today for applications in two dimensional difference gel electrophoresis (2D-DIGE) techniques. The lysine labeling CyDyes are limited by problems with over labeling resulting in protein precipitation and isoelectric point (pI) drift at high pH's. These limitations have been addressed by a family of highly water soluble and pI balancing zwitterionic BODIPY dyes, which were previously synthesized in the Grieco group. The absorbance maxima of the BODIPY fluorophores were tuned through extension of the pi system to produce a three color, spectrally resolved dye set. However the fluorescence of the green emitting BOPIDY suffered at pH's less than 3.5 and greater than 11, while the red emitting BODIPY was susceptible to Michael addition changing its emission profile. To address the limitations of the BODIPY family of dyes, a new family of zwitterionic 2DDIGE dyes based on the established CyDye fluorophores have been synthesized. A complete three dye zwitterionic minimal labeling set which features a cysteic acid motif, titratable amine functionality and an NHS activated ester group reactive towards lysine residues has been synthesized: Z-Cy2 (QY= 6.8% ± 0.1, epsilon= 155,000), Z-Cy3 (QY= 11.1% ± 0.4, epsilon= 124,500), Z-Cy5 (QY= 43.3% ± 0.6, epsilon= 217,600). In addition, a complete three dye zwitterionic saturation labeling set which incorporates a cysteic acid motif and maleimide functionality reactive towards cysteine residues has also been synthesized: Z-Cy2-Mal (QY= 6.6 % ± 0.1, epsilon= 104,500), Z-Cy3-Mal (QY= 12.4 % ± 0.5, epsilon= 127,700), Z-Cy5-Mal (QY= 40.2 % ± 0.4, epsilon= 217,400).Item Type-2 diabetes and innate immunity : new connections revealed by multi-dimensional fractionation of blood plasma prior to proteomic analysis(Montana State University - Bozeman, College of Letters & Science, 2010) Laffoon, Scott Bradley; Chairperson, Graduate Committee: Edward DratzWe compared levels of protein isoforms in human blood plasma from patients with newly diagnosed and untreated type-2 diabetes (T2DM) with non-diabetic controls in samples obtained from US NIH. We immunodepleted fourteen of the most abundant proteins from pooled plasma samples and separated the depleted samples into six fractions by reverse-phase liquid chromatography at 80°C. Proteins from these fractions were labeled with new high quantum yield, hydrophilic and spectrally resolved fluorescent detection dyes developed at MSU and resolved on large-format (24cm x 20cm) two-dimensional gels. By fluorescence analysis of 2D gels, using >1.4 fold change and p<0.05 acceptance criteria, we have identified five T2DM associated proteins and isoforms, including: two isoforms of zinc-alpha glycoprotein (ZAG), one isoform of serum amyloid A-1 (SAA-1) preprotein, one isoform of cysteine-rich secreted protein-3 (CRISP-3), one isoform of haptoglobin, and an A1-apolipoprotein fragment. Complement factor H related-5 (CFHR-5) is the likely identification of a sixth protein found significantly down in T2DM. Changes in the plasma levels of CRISP-3 and CFHR-5 strengthen the hypothesis that T2DM is a disease involving innate immunity. Three of these proteins are known to specifically bind to the transport protein, human serum albumin (HSA). Also, CRISP-3 is a specific and high-affinity ligand of alpha 1 beta glycoprotein, which is an HSA binder. To investigate HSA binding properties, we quantitatively measured the binding of a dye probe by HSA at neutral pH. These measurements revealed that HAS binding of the probe correlates with several metabolic parameters of central importance to the diagnosis of T2DM, including fasting plasma glucose (FPG). Therefore, this assay may reveal altered properties of HSA that could be developed for the clinical assessment of individuals' metabolic status. We sought modifications of HSA or altered cargo of HSA that may cause the difference in binding. 1D gels of plasma proteins reacted with maleimide dye showed no changed levels of the oxidation state of HSA's lone thiol, Cys-34. However, 1D blots of plasma proteins reacted with the oxidative carbonyl probe, hydrazide-biotin conjugate, and probed with luminol reactive HRP-neutravidin showed a surprising anti-correlation of HSA oxidation with hemoglobin A1c, an indicator of glycemic control.Item Design and synthesis of fluorescent dyes for use in proteomic research(Montana State University - Bozeman, College of Letters & Science, 2008) Spicka, Kevin James; Chairperson, Graduate Committee: Paul Grieco; Hien Hguyen (co-chair)Proteomics is a rapidly developing field requiring powerful new technology in order to be able to detect proteins at increasingly lower concentrations. To aid in the detection of proteins at lower concentrations, DIGE dyes, a family of spectrally resolved fluorescent dyes, are currently available to proteomic researchers for 2D gel analysis. However, the demands of protein detection dictate that dyes that are even more sensitive and versatile be created. The syntheses of highly sensitive, water soluble BODIPY fluorophore dyes are described. These dyes are proposed to have the necessary sensitivity to allow for detection of proteins in much lower concentrations, providing an improvement over current protein detection limits. The BODIPY dyes that have been synthesized are available in a variety of absorbances and emissions. While fluorescent dyes that are amine-reactive are the most popular covalently binding protein labeling markers being used in today's proteomic research, thiol-reactive fluorescent markers are gaining importance in proteomic research. Since thiol residues are less common in proteins compared to their amine counterparts, saturation labeling and quantification are more easily achieved. The syntheses of sensitive thiol- reactive fluorescent dyes are described. These syntheses allow for quick generation of thiol-reactive fluorescent markers to be used in proteomic research.Item The synthesis of fluorescent and phosphorescent dyes for biochemical application(Montana State University - Bozeman, College of Letters & Science, 2010) Sanger, Elizabeth Ann; Chairperson, Graduate Committee: Paul GriecoCurrent dyes for proteomics that are applicable to multiplexing experiments suffer from lack of sensitivity and poor water solubility. A series of tetramethyl rhodamine derivatives were developed to study the effects of substitution patterns on the photophysical properties of the fluorophores. After identifying the superior fluorophore a zwitterionic side chain with properties beneficial for two dimensional applications was coupled to the fluorophore and the photophysical properties were studied. Iridium(III) cationic fluorophores are interesting synthetic targets due to their ultraviolet absorption wavelengths and visible emission properties. A series of Iridium(III) fluorophores, some of which contain a handle for further synthetic extension, has been made to study the ligand effects on the emission properties of the dyes. Finally, some of these dyes have been synthetically modified for proteomic labeling applications.Item Investigating host response to viral infection through proteomics : a study of murine norovirus(Montana State University - Bozeman, College of Letters & Science, 2008) Furman, Linnzi Marie; Chairperson, Graduate Committee: Brian Bothner; Michele Hardy (co-chair)Norovirus causes roughly 23 million cases of foodborne illnesses in the United States each year. While this virus was characterized over 30 years ago, it remains non-cultivatable in human cells, resulting in an incomplete understanding of the host cell's response to infection. However, in 2004 murine norovirus (MNV) was found to be cultivatable in mice and has since been successfully cultured in RAW 264.7 cells. MNV has become an important model system for studying norovirus, as it is structurally and genetically similar to human norovirus. A global proteomics approach using fluorescently tagged, activity-based probes and 2D differential gel electrophoresis analysis was used to study MNV infection. Specifically, the process of cell death was investigated to determine if apoptosis, or programmed cell death, occurred in response to infection. Through the 2D differential gel analysis, 27 differentially regulated proteins were identified at 4 hours post infection, and 22 differentially regulated proteins were identified at 12 hours post infection; a strong majority of these proteins have been related to apoptosis in the literature. Using fluorescently-labeled activity-based probes and fluorimetric assays, we have monitored the activation of several caspases induced by viral infection. Infected samples show a significant increase in caspase activity over control samples within the first few hours post infection, indicating a virally induced activation of caspases. Cells were also infected in the presence of a pan-caspase inhibitor, Boc-D(OMe)-fmk, which led to caspase-independent cell death. Using propidium iodide and Hoechst staining, it was concluded that infected cells undergo necrosis in the presence of the caspase-inhibitor, while those infected in the absence of the inhibitor undergo apoptosis. From these studies it can be concluded that cells infected with MNV undergo a caspase-mediated, apoptotic cell death, while the caspase-independent cell death can be classified as necrosis. This study provides significant insight to norovirus-induced cell death.