Mountain Scholar :: Browsing by Author "Peersen, Olve, committee member"

Browsing by Author "Peersen, Olve, committee member"

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Open Access
5-hydroxymethylcytosine and endonuclease G as regulators of homologous recombination
(Colorado State University. Libraries, 2017) Vander Zanden, Crystal M., author; Ho, P. Shing, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Fisk, Nick, committee member
Homologous recombination (HR) is a necessary biological process for all living organisms, and it is especially important for repairing damaged DNA. Improper HR results in DNA damage-related diseases, notably increased likelihood of cancer when HR regulators, such as the human BRCA1 gene, are impaired. HR is also a tool for biotechnology, giving scientists the power to easily delete or mutate genes and study the effects of those modifications. Recently, the epigenetically modified nucleotide 5-hydroxymethylcytosine (5hmC) was found to regulate vertebrate HR via interaction with the protein endonuclease G (EndoG). In this dissertation, I use biochemical/biophysical methods to elucidate the interaction between 5hmC and EndoG, thus working towards understanding their roles as regulators of recombination. I find that 5hmC forms a unique hydrogen bond to stabilize Holliday junctions, the four-stranded DNA intermediate in HR. 5hmC also induces a global structure change to the junction, increasing protein access to the junction crossover and providing potential for either direct or indirect readout of 5hmC. Further connecting EndoG with recombination, we present the first evidence that EndoG preferentially binds and cleaves Holliday junction DNA, implicating a role for EndoG as a resolvase. I demonstrate that EndoG recognizes 5hmC in the junction context and observe unique cleavage products from EndoG interaction with 5hmC-junctions. These results suggest that EndoG may have a previously unrecognized junction resolvase function and, in this way, play a more direct role in recombination than simply creating double-stranded breaks in duplex DNA to initiate the HR mechanism. Finally, I present a unique structural feature of vertebrate EndoG that we hypothesize is the basis for 5hmC recognition. I present the structure of mouse EndoG and propose that a two amino acid deletion, conserved in vertebrate EndoG sequences, is associated with unraveling of an α-helix. This structural perturbation positions amino acid side chains to confer 5hmC-sensing ability to all vertebrate EndoG. I expect that these deletion mutations and resulting structural effects co-evolved with the appearance of 5hmC in vertebrate genomes to give EndoG an additional function of recognizing 5hmC in the cell. Overall this work is building onto the understanding of 5hmC and EndoG as markers and regulators of recombination.
Open Access
Analysis of virus-derived small RNAs reveals that the RNA silencing response to flavivirus infection differs dramatically between C6/36 and Aag2 mosquito cell lines
(Colorado State University. Libraries, 2010) Scott, Jaclyn Christine, author; Blair, Carol D., advisor; Olson, Kenneth Edward, committee member; Wilusz, Carol J., committee member; Peersen, Olve, committee member
The exogenous small RNA pathway has been shown to be an important antiviral defense in mosquitoes against arboviruses such as dengue virus (DENV), but little is known about how the pathway and the virus interact in the cell. The studies described in this dissertation examine the how small RNA pathways interact with DENV and a mosquito-only flavivirus, cell-fusing agent virus (CFAV), in mosquito cell cultures. Deep sequencing of virus-specific small RNAs in Aedes aegypti Aag2 cells indicates that DENV2 is targeted by the exogenous RNA interference (RNAi) pathway in this cell line, which is consistent with the DENV2-specific small RNAs seen in DENV2-infected A. aegypti mosquitoes. When the DENV2-specific small RNAs from the Aedes albopictus C6/36 cell line were analyzed, the size and polarity of the small RNAs was not consistent with the exogenous small interfering RNA (siRNA) pathway. Further molecular analysis of the C6/36 cell line indicated that it appears to lack functional Dicer2 processing of long double-stranded RNA (dsRNA). CFAV small RNAs were also discovered in the Aag2 cell line during the deep sequencing analysis. It appears that this cell line is persistently infected with this mosquito-only flavivirus, and the virus is also targeted by the exogenous siRNA pathway in the cells. Sequence comparisons between CFAV and DENV2 RNA did not show long regions of sequence identity between the two viruses, indicating that a sequence-specific mechanism for virus-derived small RNAs from one virus to interfere with replication of the other virus during dual infections seems unlikely. The C6/36 cell line was inadvertently infected with CFAV, but the CFAV-specific small RNAs in C6/36 cells did not appear to be generated from the exogenous siRNA pathway, consistent with the DENV2-specific small RNAs in this cell line. The larger sized, mostly positive sense virus-specific small RNAs found in the C6/36 cells suggest that virus infections may be targeted by another small RNA pathway (such as the piwi-interacting pathway) in this cell line. These studies provide a better understanding of the interactions of DENV2 with the mosquito antiviral RNAi pathway in infected mosquito cells and have revealed a dysfunctional RNAi pathway in the C6/36 cell line. This work also provides a basis for further studies examining the interactions between mosquito-only flaviviruses, arboviruses and the antiviral RNAi pathway.
Open Access
Applications of inorganic nanoparticles in biological electron microscopy
(Colorado State University. Libraries, 2016) Ni, Thomas Wentung, author; Ackerson, Christopher, advisor; Prieto, Amy, committee member; Finke, Richard, committee member; Peersen, Olve, committee member
Electron microscopy is an immensely powerful for imaging at the cellular level. However, many of the macromolecules of interest are difficult to image due to low electron density. There has been an immense body of work in order to visualize these macromolecules. In the past, many of the methods of visualization revolved around staining samples with heavy metals, however these stains are non-specific. In order to develop more specific methods of tagging macromolecules, there are two different methods to consider: the first being a top-down approach, in which electron dense tags, in this case inorganic nanoparticles, are given specific ligands to take advantage of different chemistries to attach these nanoparticles to macromolecules of interest. The second method is through a bottom-up approach where biomolecules are given the specific ability to form inorganic nanoparticles. Inorganic nanoparticles have been investigated with various ligands in order to enhance binding capability to macromolecules. The chief method of functionalizing these inorganic nanoparticles comes from ligand exchange; much has been studied regarding ligand exchange, but there are still many unanswered questions. Herein, we endeavor to reveal both the mechanism of exchange and the functional unit of exchange. We also report progress towards understanding an enzyme that is capable of forming inorganic nanoparticles, which could be cloned onto proteins as well. This bottom up style has been studied in several other groups; however, none of the previously reported methods have seen much use. Herein, we report a potential NADPH-dependent enzyme that forms selenium nanoparticles.
Open Access
Biogenic nanoparticles and their application in biological electron microscopy
(Colorado State University. Libraries, 2018) Nemeth, Richard S., author; Ackerson, Christopher, advisor; Yao, Tingting, committee member; Bjostad, Louis, committee member; Peersen, Olve, committee member
Interest in nanomaterials has seen a dramatic increase over the past twenty years. In recent years many have turned toward proteins to aid in developing novel materials due to the mild reaction conditions, functionalization, and novel synthetic control of the resulting inorganic structures. Proteins have the ability to direct aggregation of inorganic nanostructures, while some enzymes are able to perform oxio/reductase activity to synthesize the materials as well. These two general properties are not always mutually exclusive and the dual function of certain proteins in nanoparticle synthesis is at the core of this work. Of all the applications for biogenic nanoparticles, generating tools for biological electron microscopy is one of the most appealing. The contrast issue, specifically with in vivo biological sample in the electron microscope has drastically limited the information obtainable by this method. An ideal biogenic nanoparticle would operate analogously to GFP in optical microscopy and contain the dual function characteristics stated above. More specifically it would have to fulfill three criteria: i) reduction of a metal precursor, ii) product size control, iii) product retention. To discover such a clonable contrast tag we must deepen our understanding of biogenic nanoparticle formation in tandem with discovering and developing novel dual function enzymes. This work encapsulates both aspects necessary for the development of a successful clonable nanoparticle for biological electron microscopy. Current biogenic synthetic methods produce nanomaterials with less desirable properties than their inorganic counterparts. Conducting fundamental research and establishing a set of rules and guidelines for biogenic methods will ultimately get us closer to mimicking the control nature has already developed. This dissertation contains 3 chapters. Chapter 2 focuses on the use of protein crystals as scaffolds for nanomaterial synthesis. Herein porous protein crystals were used to control the gold nanocluster seeded growth of gold nanorods in an attempt to help establish guidelines for biogenic nucleation controlled nanomaterial synthesis. High aspect gold nanorod products were generated from within the crystal pores. Subsequent dissolving of the crystals allowed for release of these rods from their template. The following two chapters focus on metalloid reductase nanoparticle synthesis in which we have discovered and characterized a novel selenophile bacteria. Through purification and mass spectrometry we found a glutathione reductase like enzyme to be responsible for Se nanoparticle formation. A commercially available glutathione reductase from yeast was used for Se nanoparticle formation in vitro. This mechanism was characterized and the system was assessed for potential use as a clonable tag. The native enzyme was sequenced and isolated, followed by its own characterization. Our kinetic findings suggest this enzyme is the first documented metalloid reductase due to its specificity for selenium substrates. The enzymes transportability to foreign organisms demonstrates its potential use as a clonable contrast tag for electron microscopy.
Open Access
Characterization of the selective hydrolysis of branched ubiquitin chains by Uch37 and its activator Rpn13
(Colorado State University. Libraries, 2020) Hazlett, Zachary S., author; Yao, Tingting, advisor; Cohen, Robert, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Kennan, Alan, committee member
The ubiquitin (Ub) C-terminal hydrolase, Uch37, can be found associated with the 26S proteasome as well as the INO80 chromatin remodeling complex. Bound to the 26S proteasome, it assists in regulating the degradation of Ub modified proteins. The proteasomal subunit Rpn13 binds Uch37, anchors it to the proteasome 19S regulatory particle and enhances the deubiquitinating enzyme's (DUB's) catalytic activity. While the structure of the Uch37/Rpn13 complex bound to a single Ub molecule has been characterized, much still remains unknown regarding the enzyme's substrate specificity, the molecular basis for its substrate specificity, and its function in the regulation of proteasomal degradation. In this thesis we characterize the substrate specificity of Uch37 with and without its proteasomal binding partner Rpn13. By synthesizing poly-Ub chains of various linkage types and topologies and using these Ub chains in in vitro deubiquitination assays, we were able to determine that Uch37/Rpn13 selectively cleaves branched Ub chains. This provides evidence to suggest that Uch37 is the first enzyme with activity specific for branched Ub chains. Branched Ub chains have been identified endogenously and have roles connected to the regulation of nascent misfolded polypeptides, cell cycle control, and the enhancement of proteasomal degradation. The work presented here sets out to characterize the molecular mechanism of branched chain hydrolysis by Uch37 and its binding partner Rpn13, determine the kinetics of this enzymatic reaction, and establish a system for probing the function of "debranching" by Uch37 in proteasomal degradation. The conclusion of our work builds our understanding of the complex system of intracellular signaling by Ub and unveils key elements to the primary system responsible for regulating cellular protein homeostasis.
Open Access
Development and evaluation of new leprosy skin test antigens as diagnostic tools
(Colorado State University. Libraries, 2012) Rivoire, Becky Louise, author; Brennan, Patrick J., advisor; Beaty, Barry, committee member; Gonzales-Juarrero, Mercedes, committee member; Peersen, Olve, committee member
An early diagnostic test for leprosy that is adequately sensitive and specific to identify infected individuals before the onset of clinical symptoms continues to be one of the greatest needs in the field. Preclinical diagnosis would expedite the delivery of chemotherapy to patients, prevent disabilities, decrease stigma, intercept transmission, and measure the true incidence of disease. To address this pressing need, three new leprosy skin test antigens were investigated. MLSA-LAM [M. leprae soluble antigens devoid of lipoglycans, primarily lipoarabinomannan (LAM)], MLCwA (M. leprae cell wall associated antigens), and MLMA-LAM (M. leprae membrane antigens devoid of lipoglycans, primarily LAM). Two of these antigens, MLSA-LAM and MLCwA, were developed for manufacturing and testing for safety and efficacy in phase I and phase II human clinical trials. Skin test antigens were derived from M. leprae purified from experimentally infected armadillo tissues under current good manufacturing practice conditions. A skin test pilot plant was created at Colorado State University for this purpose. Quality control testing of skin test antigens included potency and stability testing in guinea pigs, safety testing in guinea pigs and mice, integrity testing by gel electrophoresis and immunoblotting, and purity testing for residual dextran, collagenase, detergent, and endotoxin. An investigational new drug (IND) application was submitted to the Food and Drug Administration (FDA) and clinical protocols with respective informed consent forms were generated. Training in good laboratory, manufacturing, and clinical practice (GLP, GMP, and GCP) was a prerequisite for these studies. The phase I clinical trial was conducted at a non-endemic region for leprosy with both antigens at 2.5, 1.0, and 0.1 µg dosages. A randomized double blind phase II clinical trial (stages A, B, and C) followed in an endemic region for leprosy with both antigens at 1.0, and 0.1 µg dosages. Antigens were tested in the phase I and phase II, stage A/B trials using the intradermal delayed type hypersensitivity (DTH) skin test in healthy subjects without known exposure to leprosy, while the phase II, stage C trial compared the DTH skin test to the IFN-γ test and the M. leprae specific phenolic glycolipid I antibody test in target populations, including: leprosy patients, household contacts of leprosy patients, and tuberculosis patients. Both skin test antigens, MLSA-LAM and MLCwA, were found to be safe at each dose tested in the phase I and II clinical trials. The phase II, stage A/B clinical trials showed the baseline in healthy endemic controls for both leprosy antigens at the low dose of 0.1 µg was negligible, while slightly elevated with the high dose of 1.0 µg. Efficacy findings from the phase II, stage C clinical trial showed that the antigens were immunologically potent; highly specific, but lacked sensitivity at the low dose. The response to PPD did not correlate with either leprosy antigen at either dose. The IFN-γ release test provided the best diagnostic accuracy at the high dose with both antigens. Household contacts with the highest risk of infection reacted in each test. MLSA-LAM and MLCwA are the first skin test antigens to show specificity for leprosy in the field. The interferon gamma release assay with MLSA-LAM at the high dose provides the best diagnostic accuracy for tuberculoid leprosy patients. The PGL-I antibody assay provides the best diagnostic accuracy for lepromatous leprosy patients. Optimization of the antigen dosage or use of these tests in parallel or combination could lead to enhanced sensitivity, resulting in a good early diagnostic test for leprosy. Results from these research studies provide proof that a product can be translated from the bench to the clinic in an academic setting.
Open Access
Engineering stabilized enzymes via computational design and immobilization
(Colorado State University. Libraries, 2016) Johnson, Lucas B., author; Snow, Christopher, advisor; Reardon, Kenneth, committee member; Peebles, Christie, committee member; Peersen, Olve, committee member
The realm of biocatalysis has significantly matured beyond ancient fermentation techniques to accommodate the demand for modern day products. Enzymatically produced goods already influence our daily lives, from sweeteners and laundry detergent to blood pressure medication and antibiotics. Protein engineering has been a major driving force behind this biorevolution, yielding catalysts that can transform non-native substrates and withstand harsh industrial conditions. Although successful in many regards, computational design efforts are still limited by the crude approximations employed in searching a complex energy landscape. Advancements in protein engineering methods will be necessary to develop our understanding of biomolecules and accelerate the next generation of biotechnology applications. Our work employs a combination of computational design and simulation to achieve improved enzyme stability. In the first example, an enzyme used in the production of cellulosic biofuels was redesigned to remain active at high temperature. An initial approach involving consensus sequence analysis, predicted point mutation energy, and combinatorial optimization resulted in a sequence with reduced stability and activity. However, by using recombination methods and molecular dynamics simulations, we were able to identify specific mutations that had a stabilizing or destabilizing effect, and we successfully isolated mutations that benefited enzyme stability. Our iterative approach demonstrated how common design failures could be overcome by careful interpretation and suggested methods for improving future computational design efforts. In the second example, a cellulase was designed to have a high net charge via selected surface mutagenesis. “Supercharged” cellulases were experimentally characterized in various ionic liquids to assess the effect of high ion concentration on enzyme stability and activity. The designed enzymes also provided an opportunity to systematically probe the protein-solvent interface. Molecular dynamics simulations showed how ions influenced protein behavior by inducing minor unfolding events or by physically blocking the active site. Contradictory to previous reports, charged mutations only appeared to alter the affinity of anions and did not significantly change the binding of cations at the protein surface. Understanding the different modes of enzyme inactivation could motivate targeted design strategies for engineering protein resilience in ionic solvents. In addition to the discussed computational design methods, immobilization strategies were identified for capturing enzymes within porous protein crystals. Immobilization offers a generic approach for improving enzyme stability and activity. Our preliminary studies involving horseradish peroxidase and other enzymes suggested protein scaffolds could be employed as an effective immobilization material. Co-immobilizing multiple enzymes within the porous material led to improved product yield via exclusion of off-pathway reactions. Although future studies will be required to assess the potential capabilities of this immobilization strategy in comparison to other materials, preliminary results suggest protein crystals offer a favorable, controlled environment for immobilizing enzymes. The diversity of approaches presented in this thesis emphasizes that there are many options for engineering enzyme stability. Extending the lessons learned from our cellulase engineering to the greater field of rational protein design promotes the concept of biomolecules as designable entities. By establishing the shortcomings of our designs and suggesting routes for improvement, we anticipate our design methods and immobilization strategies will procure continued interest from the biotechnology community. The toolsets we developed for cellulases can be directly transferred to other enzymes and have the potential to impact a range of protein engineering applications.
Open Access
Factors and mechanisms of archaeal transcription termination and DNA repair
(Colorado State University. Libraries, 2022) Marshall, Craig, author; Santangelo, Thomas J., advisor; Peersen, Olve, committee member; Wilusz, Carol, committee member; Yao, Tingting, committee member
RNA synthesis by RNA polymerase (RNAP) is an essential process and must be properly regulated both temporally and spatially to ensure cellular health in dynamic environments. Regulation of RNA synthesis in response to internal and environmental stimuli is typically achieved through interactions with RNAP at all stages of the transcription cycle- initiation, elongation, and termination. While studies of transcription initiation and elongation have identified multiple regulatory transcription factors and defined mechanisms, only a handful of protein factors able to terminate transcription have yet been described, and the general mechanism of transcription termination is still highly debated. We previously identified the first two factors capable of terminating transcription elongation complexes (TECs) in Archaea from the genetically tractable Thermococcus kodakarensis, and use both factors as models to explore the molecular mechanisms involved in collapse of the TEC. The Factor that terminates transcription in Archaea (FttA), a close homolog of the human CPSF subunit CPSF73, is completely conserved throughout Archaea, and appears to act analogously to the bacterial termination factor Rho, terminating transcription after the uncoupling of transcription and translation at the end of protein coding genes. We employed a novel genetic screen to verify the role of FttA in the polar repression of transcription, a phenomenon specific to regulation of genes contained within operons in prokaryotes. Eta, a euryarchaeal-specific superfamily 2 (SF2) helicase, appears to terminate transcription in a more specialized context, potentially terminating transcription of TECs arrested at sites of DNA damage while concurrently recruiting appropriate DNA repair enzymes, akin to the bacterial termination factor Mfd. A structure-function study of Eta employing select mutations derived from a crystallographic structure was conducted to elucidate the Eta-TEC contacts and various activities of Eta required for Eta-mediated termination. Further, many efforts were directed at establishing a role of Eta as an archaeal transcription-repair coupling factor (TRCF), and while this was not achieved, a state-of-the-art next-generation sequencing based approach to monitor nucleotide excision repair (NER) and the sub pathway transcription-coupled repair (TCR) genome-wide was developed and verified in E.coli. The work in this dissertation adds valuable insight to multiple fields of research. First, exploration into the mechanism of Eta-mediated transcription termination reveals a potential shared susceptibility of core RNAP subunits to transcription termination while elucidating activities of SF2 helicases- enzymes which are ubiquitously distributed in multiple essential cellular pathways. Second, our genetic screen identifies FttA as the archaeal polarity factor, shedding light on functions of an ancestral factor indispensable in mammalian transcription termination pathways. Establishment of the novel RADAR-seq/RNA-seq measurement of NER genome-wide will likely prove instrumental in future studies of archaeal DNA repair, and potentially presents a new paradigm in research of eukaryotic-like NER by use of Archaea as a advantageous model organism.
Open Access
Fundamental research into gold nanocluster properties
(Colorado State University. Libraries, 2021) Window, Phillip S., author; Ackerson, Christopher J., advisor; Neilson, James R., committee member; Kennan, Alan J., committee member; Peersen, Olve, committee member
Gold materials are popular for research into many applications with their interesting properties, such as magnetism, bio-inactivity, and other size-dependent properties. As the size of the gold material decreases from a bulk material to the nanoscale, new properties are introduced moving through different size regimes. As the particle size reaches the 2-3 nm range and move into the quantum-confined particle range, the most interesting particle changes occur and gold nanomaterials have extremely interesting research potential. These materials exist between the bulk and molecular systems and have similar properties to both; however, they are different enough from both of these to have their own unique application possibilities. Some properties of gold nanoclusters can be attributed more to the core or more to the ligand layer of the nanocluster. Certain properties, like electronics and magnetism, are due to the superatomic electron count and electronic structure from the core and depend on the number of gold atoms in the nanocluster. Extensive research has been done on investigating and altering these properties in small nanoclusters, however, larger nanoclusters have hardly been studied as they can be more difficult to work with. Within this work is investigated the magnetism and thus electronic structure of Au102(SPh)44 and Au133(tBBT)52 in different oxidation states. Paramagnetism up to two unpaired electrons is observed with both these nanoclusters through solution phase magnetic studies. Through this, electronic structure information has been obtained to elucidate the behavior of unique superatomic 1G and 1H orbitals. Looking at the outside of a nanocluster structure, interactions of nanoclusters with other nanoclusters, molecules, surfaces, and solvents are all due to the ligand layer of the nanocluster. Investigations of the ligand layer have been performed extensively through many techniques. However, further studies are always helpful since controlling the ligand layer is essential for functionalization for potential applications. Within this work is investigated the interactions of Au25(SR)18 with other Au25 nanoclusters in both solution and solid phase, as well as ligand exchange reactions of Au133(tBBT)52. Studies on Au25(SR)18 within solution include investigations of a supramolecular assembly, or supercluster, formed solely of the nanocluster itself with control over its growth and size. Studies on Au25(SR)18 within the solid-phase include controlled crystallization techniques that result in different solid-phase structures with previously unseen properties. Ligand exchange studies have also been expanded from small nanocluster materials only in previously published studies to the large nanocluster, Au133(tBBT)52. Within this dissertation, some of the first empirical studies into the oxidation state- dependent properties of large gold nanoclusters, Au102(SPh)44 and Au133(tBBT)52, were performed. This betters the field's understanding of how many unpaired electron spins these large gold nanocluster can sustain at room temperature and further elucidates the behavior of superatomic electronic structure and behavior based on electron count. Furthermore, this dissertation presents the first investigations into the formation of supramolecular assemblies of gold nanocluster as recyclable materials, and more interactions of gold nanoclusters based on ligand layer interactions through polymorphism studies and ligand exchange studies. These investigations all help understand how to control the ligand layer for future applications of gold nanoclusters and nanoparticles, from molecular to bulk materials.
Open Access
Generation of site-specific ubiquitinated histones through chemical ligation and characterization of histone deubiquitinases
(Colorado State University. Libraries, 2016) Al-afaleq, Nouf Omar, author; Yao, Tingting, advisor; Cohen, Bob, committee member; Fisk, Nick, committee member; Peersen, Olve, committee member
Nucleosome is the basic unit of chromatin and is composed of 147 base pairs of DNA wrapped 1.65 turns around a histone octamer of the four core histones (H2A, H2B, H3 and H4)(Luger et al., 1997). Histones are subject to numerous post-translational modifications. One such modification is the addition of a single ubiquitin (Ub) moiety to a specific lysine residue in the histones, such as H2AK119 or H2BK120 in humans. Depending on the site of Ub attachment, these modifications have distinct functional consequences. Whereas H2A ubiquitination is associated with transcriptional repression and silencing, H2B ubiquitination is associated with actively transcribed regions and has roles in initiation, elongation and mRNA processing. A more recently discovered ubiquitination site in H2A, H2AK13/15, is associated with DNA damage repair. In addition, a number of other ubiquitination sites on all types of histones have been discovered by high throughput mass spectrometry. The functions and regulations of those novel ubiquitinations are not known. Deubiquitinating enzymes (DUBs) reverse these ubiquitinations and therefore, are involved in a variety of regulatory processes. Mutations in several histone DUBs have been implicated in various diseases, thus they represent potential therapeutic targets. The specificity and regulation of histone DUBs are poorly understood in part because it has been difficult to obtain homogenous ubiquitinated histones and nucleosomes to use as substrates in vitro. Previously, several strategies have been developed to produce chemically defined ubiquitinated histones that use a combination of expressed protein ligation (EPL) and solid phase peptide synthesis (SPPS) techniques. These protocols are technically challenging for a biochemical lab. This dissertation describes our successful approach in obtaining homogenous site-specific ubiquitinated H2A and H2B that were then reconstituted into nucleosomes and used to qualitatively and quantitatively characterize a panel of known histone DUBs in vitro. We anticipate that our approach can be applied to generate all types of Ub-histone conjugates regardless of the particular ubiquitination site or histone types. They will significantly facilitate the study of all types of histone ubiquitination.
Open Access
Halogen bonds in biological macromolecules
(Colorado State University. Libraries, 2016) Scholfield, Matthew Robert, author; Ho, P. Shing, advisor; Fisk, John, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member
The purpose of this dissertation is to study how halogen bonds (X-bonds) affect the stability of biological macromolecules and to develop a set of empirical mathematical equations that can provide insight into the anisotropic nature of covalently bound halogens. To achieve this end, we first conducted a detailed analysis of the Protein Data Bank (PDB) to determine the prevalence of X-bonding in biological macromolecules, which allowed us to study the geometrical trends associated with X-bonding. Quantum mechanical (QM) calculations were also applied to determine how the strength of X-bonds interaction could be "tuned." The next chapter used QM calculations to help parameterize an equation that can model the anisotropic size and charge of covalently bound chlorine, bromine and iodine. The energies obtained from this equation were validated on experimentally determined X-bond data by differential scanning calorimetry (DSC) in DNA holiday junctions and were found to nearly duplicate the energies obtained in the solution state experiments. In the final chapter, we engineer X-bonds into the structure of T4 lysozyme to studying structural and thermodynamic effects of X-bonds on protein. X-bonds were introduced into the enzyme via site-specific non-canonical amino acid incorporation and then the structure and stability of the protein were assayed via X-ray crystallography and DSC, respectively. The culmination of this work has elucidated many concepts that need to be considered when trying to engineer new biologically based materials with halogens.
Open Access
Inhibition of a truncated form of human mitochondrial kidney-type glutaminase (hKGA124-551) by bis-2-(5-phenylactamido-1,2,4-thialdiazol-2-yl)ethyl sulfide (BPTES)
(Colorado State University. Libraries, 2011) Hartwick, Erik William, author; Curthoys, Norman, advisor; Ho, P. Shing, committee member; Peersen, Olve, committee member; Mykles, Donald, committee member
Mitochondrial glutaminase (GA) catalyzes the hydrolysis of glutamine producing glutamate and an ammonium ion. There are three isoforms of mammalian GA that are essential to hepatic ureagenesis, renal ammoniagenesis, synthesis of the neurotransmitter glutamate, and the catabolism of glutamine. Here we focus on the human KGA isoform that is predominantly expressed in kidney, brain, intestine, and tissues of the immune system. Recent publications suggest that GA is a novel target for developing new cancer therapeutics. These studies have indicated that inhibition of GA by small molecule inhibitors significantly reduces the size of tumors in rats and inhibits growth of transformed cells in culture. A truncated form of human KGA hKGA124-551 that contains amino acids 124-551, was produced to delete the C-terminal sequences that are unique to the KGA and GAC isoforms. This construct was assayed in the presence of (bis-2-(5-phenylactamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). BPTES is a potent small molecule inhibitor of mammalian GA that was previously shown to inhibit rat KGA in µM concentrations. In the current study, we adapted the standard GA assay to a microtiter plate format and used it to characterize the inhibition of hKGA124-551 using µM amounts of BPTES. Our data indicate that BPTES is a mixed non-competitive inhibitor at low concentrations of phosphate, but at higher phosphate concentrations the inhibition is predominantly uncompetitive. Lastly, gel filtration and dynamic light scattering experiments were performed to determine if hKGA124-551 oligomers are formed in the presence of BPTES and to characterize the effect of increasing concentrations of phosphate. The data suggest that in low phosphate and in the absence of BPTES, the hKGA124-551 exists as a dimer, but in the presence of BPTES and higher phosphate concentrations the molecular weight shifts to a tetramer or higher oligomer. The combined data indicate that BPTES is a potent lead compound for the development of a therapeutic inhibitor of human GA that may be a potential cancer therapeutic.
Open Access
Investigating the roles prion-like domains play in cellular stress responses
(Colorado State University. Libraries, 2018) Shattuck, Jenifer Elizabeth, author; Ross, Eric, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Telling, Glenn, committee member
Prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates play an important role in regulating a broad-range of cellular functions. In the budding yeast Saccharomyces cerevisiae, at least 10 proteins have been identified that form self-propagating amyloid-based prions. Most known yeast prion proteins contain a low-complexity, intrinsically-disordered prion-forming domain that is converted into stable, detergent-insoluble aggregates, necessary for prion activity. These prion-forming domains tend to be glutamine/asparagine (Q/N) rich, and relatively lacking in charged and hydrophobic amino acids. To better understand the amino acid sequence features that promote prion activity, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains (PrLD). While from this study we did not identify new yeast prion proteins, we identified several PrLDs with aggregation activity. Therefore, in follow up studies we investigated the role these PrLDs play in other protein assemblies involved in cellular stress responses. First, we investigated how a prion-like protein kinase, Sky1, plays a role in regulating stress granules. Stress granules are cytoplasmic assemblies that form when translation initiation is limiting, including under a variety of stress conditions. Because these cytoplasmic granules are important regulatory machinery for cellular homeostasis, mutations that increase stress granule formation or decrease clearance have been linked to various neurodegenerative diseases. We provided evidence that Sky1 is recruited to stress granules through its aggregation-prone PrLD, and it phosphorylates an RNA-binding protein to efficiently disassemble stress granules. Additionally, we showed when Sky1 is overexpressed it can compensate for defects in other disassembly pathways. These findings contribute to understanding the regulation of stress granules, and provides a possible mechanism to mitigate persistent stress granules in neurodegenerative diseases. Next, we investigated how PrLDs are used to assemble and activate a vacuole-signaling complex. Many cellular processes are regulated primarily through the production of phosphoinositides. Specifically, synthesis and turnover of phosphatidylinositol 3,5 bisphosphate (PtdIns(3,5)P2) is regulated by a vacuole-signaling complex, containing prion-like proteins Fab1, Vac7, and Vac14. Interestingly, during hyperosmotic stress, there is a rapid and dramatic rise in PtdIns(3,5)P2, which leads to vacuole remodeling, critical for cellular survival. We used aggregation-altering mutations to characterize the role of Fab1's PrLD in response to osmotic stress. Overall, these studies provided evidence that Fab1's activation requires its aggregation prone PrLD for recruitment and efficient activation for cellular adaptation to stress. Collectively, the studies described below provide insights into the diverse roles PrLDs play in regulating cellular stress responses. Moreover, these studies have contributed to the field of aggregation-mediated cellular regulation by identifying new proteins involved, new proposed mechanisms, and new insights into the cellular consequences that arise from perturbations in regulation of these processes.
Open Access
Mediation of kinetochore-microtubule interactions through the Ndc80 complex component Hec1
(Colorado State University. Libraries, 2011) Guimaraes, Geoffry J., author; DeLuca, Jennifer, advisor; Bamburg, James, committee member; Peersen, Olve, committee member; Reddy, A. S. N., committee member
To view the abstract, please see the full text of the document.
Open Access
Methods for detecting and developing protein-protein or protein-RNA interactions
(Colorado State University. Libraries, 2014) Blakeley, Brett D., author; McNaughton, Brian, advisor; Kennan, Alan, committee member; Fisk, Nick, committee member; Reynolds, Melissa, committee member; Peersen, Olve, committee member
Potent and selective recognition of disease-relevant macromolecules - such as proteins and RNA - is the molecular basis of most pharmaceuticals . Historically, small (< 500 Da) molecules have filled this role. However, the overwhelming majority (~85%) of the proteome - and emerging therapeutic targets such as RNA - present a serious challenge to small molecule-dependent recognition. An alternative approach to potent and selective recognition and regulation of disease-relevant proteins and RNA is to use synthetic proteins. In contrast to small molecules, the size, relatively high folding energies (>10 kcal/mol) and functional group diversity (by virtue of proteinaceous amino acids) allow proteins to recognize - and potentially control - macromolecular receptors that evade small molecules. Presented here are two approaches to advancing the discovery of new proteins that recognize either disease-relevant protein or RNA targets. The first part of this thesis describes split superpositive GFP reassembly as a method to identify novel protein-protein interacting pairs in living cells (E. coli). The second part of this thesis describes basic studies to evaluate the suitability of a naturally occurring RNA Recognition Motif (RRM) as a scaffold for targeting disease-relevant RNA hairpins, and the development of new RRMs that target TAR RNA, a hairpin critical to HIV proliferation.
Open Access
Optimizing a synthetic signaling system, using mathematical modeling to direct experimental work
(Colorado State University. Libraries, 2014) Havens, Keira, author; Medford, June, advisor; Prasad, Ashok, advisor; Antunes, Mauricio, committee member; Peersen, Olve, committee member
Synthetic biology uses engineering principles and biological parts to probe existing biological networks and build new biological systems. As biological components become better characterized, synthetic biology can make use of predictive mathematical models to analyze the activity of biological systems. This thesis demonstrates the utility of modeling in optimizing a synthetic signaling system for a bacterial testing platform and advances the use of model-based bacterial systems as an effective tool of plant synthetic biology. Using models in combination with experimental data, I showed that increasing the concentration of a single component of the synthetic signaling system, the PBP, results in a 100 fold increase in sensitivity, and an order of magnitude increase in fold change response in the response of the bacterial testing platform. Additional mathematical exploration of the system identified another component, the number of PhoB inducible promoters, which could be adjusted to further increase maximum signal. In addition, our model has suggested additional avenues of research, including the potential to introduce new functions, such as memory, to the existing circuit. In this way the prototype synthetic signaling system developed by the Medford Lab has been refined to improve detection and generate substantial response, moving the technology closer to real-world use. Once validated, this modeling based protocol, using a microbial platform for developing and optimizing plant synthetic systems, will serve as a foundation for engineering advanced plant synthetic systems.
Open Access
Pharmacokinetic and pharmacodynamic evaluation of HIV-1 pre-exposure prophylaxis candidates in humanized mice
(Colorado State University. Libraries, 2014) Veselinovic, Milena, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Gonzalez-Juarrero, Mercedes, committee member; Peersen, Olve, committee member
In the absence of a vaccine, alternative preventative approaches against HIV-1 are needed. In pre-exposure prophylaxis (PrEP) approach, antiretroviral drugs, broadly neutralizing antibodies (bnAb) or other biological molecules are administered orally or topically for the prevention of HIV-1 infection. For successful PrEP design pharmacokinetic (PK) and pharmacodynamic (PD) studies are needed to define protective levels of antiretrovirals in mucosal tissues. The RAG-hu mice used here represent a small animal model in which human immune system is reconstituted by haematopoietic stem cells (HSC) in the immunodefficient BALB/c- Rag1-/- γc-/- and BALB/c- Rag2-/- γc-/- mice. This model was previously shown to be suitable for HIV-1 mucosal transmission and protection studies. In the experiments presented here we evaluated the utility of RAG-hu mice for the study of PK-PD aspects of antiretroviral drugs in the context of PrEP. The PK studies focused on tissue distribution of the RT inhibitor Tenofovir (TFV), the integrase inhibitor Raltegravir (RAL) and the entry inhibitor Maraviroc (MVC) following single and combinatorial oral application. Drug kinetics were examined systemically in blood plasma, and in vaginal, rectal and colonic mucosal tissues, which are the sites of HIV-1 transmission and initial viral spread. Antiretrovirals were applied in human equivalent doses to achieve steady state kinetics. Data obtained from single oral applications verified favorable PrEP profile of TFV. While results showed that RAL and MVC represent promising PrEP candidates, the data suggest that the PrEP doses would need to be higher than therapeutic ones in order to allow for once a day dosing. In combinatorial TFV/RAL and TFV/MVC oral application studies, increase in the active form of TFV (Tenofovir diphosphate, TFV-DP) accompanied by agonistic effect for the second drug in combination was observed, which can be characterized as highly favorable for PrEP applications. This is the first report on combinatorial PK of TFV, RAL and MVC in mucosal tissues which informs further testing of TFV/MVC and TFV/RAL PrEP approaches in non-human primates (NHP) and in clinical settings. For topical PrEP potential, PK profiles of TFV, RAL and MVC were also evaluated in vaginal mucosa following topical application of gel formulations. With all three drugs, one to two log higher concentrations were achieved in vaginal mucosa compared to oral application reflecting previous findings in humans. Intracellular concentrations of TFV-DP in humanized mice corresponded to the levels observed previously in human vaginal mucosa. In PD studies, the protective effect of topical PrEP with single drug and combinatorial TFV, RAL and the RT inhibitor UC781 gels was evaluated against mucosal HIV-1 transmission. High level of protection was seen with combinatorial microbicide gels - 80% (4/5) protection by TFV/UC781 gel and 87.5% (7/8) protection by TFV/RAL gel, indicating their suitability for further testing in preclinical trials. In another PD study, protective efficacy of bnAb VRC01 was examined against mucosal transmission of HIV-1 in the form of topical PrEP. The VRC01 gel (1 mg/ml) conferred protection in 77.7% (7/9) animals, while the combination of b12, 4E10, 2F5 and 2G12 bnAb which target different epitopes on the HIV-1 envelope conferred complete protection (5/5 animals) against HIV-1 mucosal transmission. These data suggest that bnAb could be effective agents for topical PrEP against HIV-1. In summary, these proof of concept PK and PD studies validated RAG-hu mouse model for preclinical evaluation of new anti-HIV-1 drugs and bnAb for oral and topical PrEP, thus providing data for further NHP studies and human clinical trials.
Open Access
Protein resurfacing to identify macromolecular assemblies
(Colorado State University. Libraries, 2016) Chapman, Alex Michael, author; McNaughton, Brian, advisor; Van Orden, Alan, committee member; Rovis, Tomislav, committee member; Fisk, Nick, committee member; Peersen, Olve, committee member
Protein engineering is an emerging discipline that dovetails modern molecular biology techniques with high-throughput screening, laboratory evolution technologies, and computational approaches to modify sequence, structure, and in some cases, function and properties of proteins. The ultimate goal is to develop new proteins with improved or designer functions for use in biotechnology, medicine and basic research. One way to engineer proteins is to change their solvent exposed regions through focused or random 'protein resurfacing'. Here, I describe several approaches towards the development of synthetic proteins with new properties and function, including resistance to aggregation, increased solubility, and potent and selective macromolecule recognition. The first part of this thesis describes the use of protein supercharging to develop a split-superpositive GFP reassembly assay that is more efficient, faster, and more robust than previously described variants, largely due to increased resistance to aggregation. The second part of this thesis describes the use of shape complementarity, protein resurfacing, and high-throughput screening to evolve the first potent and selective protein-based inhibitor of the oncoprotein gankyrin. Concomitant with this work, I also describe a protein grafting strategy to identify a soluble mimic of S6 ATPase, which is subsequently used to characterize the S6 ATPase/gankyrin interaction by isothermal titration calorimetry.
Open Access
Proteomic profiling of the rat renal proximal convoluted tubule in response to chronic metabolic acidosis
(Colorado State University. Libraries, 2013) Freund, Dana Marie, author; Curthoys, Norman, advisor; Prenni, Jessica, advisor; Nyborg, Jennifer, committee member; Peersen, Olve, committee member; Dobos, Karen, committee member
The human kidneys contain more than one million glomeruli which filter nearly 200 liters of plasma per day. The proximal tubule is the segment of the nephron that immediately follows the glomeruli. This portion of the nephron contributes to fluid, electrolyte and nutrient homeostasis by reabsorbing 60-70% of the filtered water and NaCl and an even greater proportion of NaHCO3. The initial or convoluted portion of the proximal tubule reabsorbs nearly all of the nutrients in the glomerular filtrate and is the site of active secretion and many of the metabolic functions of the kidney. For example, the proximal convoluted tubule is the primary site of renal ammoniagenesis and gluconeogenesis, processes that are significantly activated during metabolic acidosis. Metabolic acidosis is a common clinical condition that is characterized by a decrease in blood pH and bicarbonate concentration. Metabolic acidosis also occurs frequently as a secondary complication, which adversely affects the outcome of patients with various life-threatening conditions. This type of acidosis can occur acutely, lasting for a few hours to a day, or as a chronic condition where acid-base balance is not fully restored. Chronic metabolic acidosis, where the decrease in blood pH and bicarbonate last for 7 days, was the main focus of these studies. Acid-base homeostasis is achieved, in part, by the reabsorption of bicarbonate and excretion of ammonium ions and acids by the proximal convoluted tubule. Metabolic acidosis is partially compensated by an adaptive increase in renal ammoniagenesis and bicarbonate synthesis. During acidosis, there is increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule. This process generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. This response is mediated by a pronounced remodeling of the proteome of the proximal convoluted tubule that also produces an extensive hypertrophy. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. Here, a workflow was developed to globally characterize the mitochondrial proteome of the proximal convoluted tubule. Two-dimensional liquid chromatography coupled with mass spectrometry (2D/LC-MS/MS) was utilized to compare mitochondrial enriched samples from control and chronic acidotic rats. Label-free quantitative strategies are commonly used in shot-gun proteomics to detect differences in protein abundance between biological sample groups. In this study we employed a combination of two such approaches, spectral counting (SpC) and average MS/MS total ion current (MS2 TIC). In total, forty nine proteins were observed to be significantly altered in response to metabolic acidosis (p-value < 0.05). Of these, 32 proteins were uniquely observed as significantly different by SpC, 14 by MS2 TIC, and only 3 by both approaches. Western blot analysis was used to validate the fold changes of eight of the proteins that showed an increase upon acidosis. Furthermore, using an antibody specific to acetylated lysine modifications indicated that chronic acidosis causes a 2.5 fold increase in this modification specifically in mitochondria. Western blot analysis established that the observed alterations in both protein abundance and lysine acetylation are not due to the associated hypertrophy. This study represents the first comprehensive analysis of whole mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis. Additionally, our analysis demonstrates an innovative dual approach for protein quantitation. To further our understanding of the impact of acidosis on the mitochondrial proteome, mitochondrial inner membranes were isolated from control and acidotic rat proximal convoluted tubules. Additional LC-MS/MS analysis was performed, representing the first proteomic characterization of the mitochondrial inner membrane proteome of the rat renal proximal convoluted tubule. Specific sites of lysine acetylation were identified both in the inner membrane and whole mitochondria, the majority of which are novel sites. The results presented here showed successful enrichment of mitochondrial inner membranes and described the proteins and the known biological processes of this compartment of the mitochondria. Previous proteomic analysis was performed on brush-border membrane vesicles isolated from proximal convoluted tubules from control, 1 d and 7 d acidotic rats. To validate the observed protein alterations, western blot analysis was performed on freshly isolated apical membrane. Additionally, the results from three independent proteomic studies focused on the apical membrane, mitochondrial, and soluble cytosolic fractions of the proximal convoluted tubules were compiled. Bioinformatics analysis was performed to describe predominate cellular processes and pathways that respond to chronic metabolic acidosis. The results of these studies demonstrate that the physiological response to the onset of metabolic acidosis requires pronounced changes in the renal proteome. The observed proteomic adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH4+ and HCO3- ions. Overall, this dissertation describes the profiling of the proximal convoluted tubule proteome in response to chronic metabolic acidosis and provides the framework for future studies.
Open Access
Quantifying ubiquitin dynamics
(Colorado State University. Libraries, 2019) Bollinger, Sarah A., author; Cohen, Robert E., advisor; Peersen, Olve, committee member; Yao, Tingting, committee member; Prenni, Jessica, committee member; Alan, Kennan, committee member
Ubiquitin (Ub) is a small protein that is frequently attached to other proteins as a post-translational modification (PTM) to elicit a new function, cellular localization, or otherwise modulate the activity of the substrate protein. Ub addition and removal serves as a signal for proteasome degradation, regulation of cell division, gene expression, membrane and protein trafficking and signaling in a multitude of stress response mechanisms. Defects in ubiquitination or deubiquitination have been linked to cancer onset and progression, muscle dystrophies, and disorders in inflammation and immunity; these findings further highlight the critical processes regulated by Ub. Due to its high demand, cellular Ub levels are highly regulated, such that the abundance of free Ub is above a threshold enabling new ubiquitination events, a critical part of normal cell function and survival. Due to the high demand on the cellular free Ub pool to supply substrate for thousands of ubiquitination reactions, it is tightly regulated in many ways. Our knowledge of Ub homeostasis has not advanced, likely due to the lack of accurate, sensitive methods for pool quantitation that can be performed routinely. Here, a method is presented that utilizes a high affinity free Ub binding protein to quantify cellular pools of Ub after a series of treatment protocols. The methods can be performed within a day and are amenable to high throughput applications. Using these methods, the Ub pool distributions of cells under conditions such as proteasome inhibitor and heat stress were assessed. However, this assay will only report the steady-state concentration of Ub in each pool; it provides no information about the rate of movement through them. The rates of competing ubiquitination and deubiquitination or degradation reactions determine the steady-state level of every Ub-protein conjugate; however, measurement of the rate of Ub movement these conjugates remains a challenge. Thus, the relative contributions of conjugation and disassembly rates in cellular responses to different signals are rarely known. Moreover, even though the concentration of a particular Ub-protein conjugate may appear unchanged, the flux of Ub through that conjugate might change dramatically. To address these deficits in our understanding of ubiquitination, we have developed a method to label Ub and follow its movement through conjugation pathways that we call SILOW or Stable Isotope Labeling with ¹⁸O-Water. Our method is applicable to both yeast and mammalian cells, does not perturb cellular physiology in any way and can be used with conventional proteomics methods. SILOW permits rapid changes in Ub flux to be evaluated over short times across hundreds of sites within the human cell proteome to reveal the intracellular dynamics of Ub-conjugation in specific Ub-Ub linkages of polyUb compared with Ub-protein linkages of histones.