Theses and Dissertations

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Open Access
Aromatic circular dichroism in globular proteins: applications to protein structure and folding
(Colorado State University. Libraries, 1994) Grishina, Irina Borisovna, author; Woody, Robert W., advisor
The exciton couplet approach was applied to estimate the circular dichroism (CD) of Trp side-chains in proteins. Calculations were performed by the origin-independent version of the matrix method, either for the indole Bb transition only or for the six lowest energy indole transitions. The dependence of the CD of a Trp pair upon its distance and geometry has been analyzed. It was predicted that mixing with far-uv transitions are as important in determining the CD intensity of the near-uv transitions as the coupling among near-uv transition. The effects of varying exposure of Trp chromophores and nearby charges on Trp CD have been examined. A survey of a large number of proteins from the Protein Data Bank reveals a number of cases where readily detectable exciton couplets are predicted to result from the exciton coupling of Trp Bb bands. The predicted CD spectra are generally couplets, often dominated by the contributions of the closest pair, but sometimes exhibit three distinct maxima. This CD depends on the distance and relative orientation of Trp pairs and thus reflects the spatial arrangement of Trp residues in the protein. It was shown that Trp side chains can make significant contributions to the CD of proteins in the far ultraviolet. The distance dependence of exciton splitting, rotational and couplet strengths of Trp pairs show general agreement with theoretical predictions. In several cases, changes in protein Trp CD can be attributed to a specific Trp pair and explained as a definite change in its conformation. Applications of the exciton couplet approach are discussed for various crystal forms of hen lysozyme, turkey and human lysozyme. Trp62 in hen and turkey lysozymes was found to be sensitive to the perturbations of the protein surface due to binding of substrate, antibodies and intermolecular contacts in the crystal. Conformational changes of Trp62 are predicted to have a strong effect on the overall Trp CD of lysozyme. Predicted Trp CD is compared with experimental results for various lysozymes, a-chymotrypsin and chymotrypsinogen A, concanavalin, dihydrofolate reductase and ribonuclease from Bacillus intermedius 7P (binase). The calculated near-uv CD for hen lysozyme matches the experimental amplitude. Correlation of conformational changes in proteins with Trp CD is shown for a-chymotrypsin and chymotrypsinogen A. We found that the exciton couplet approach might be useful in relating Trp CD and changes in protein structure upon local mutations and conformational changes involved in enzyme activation. Small globular proteins are usually composed of a single structural domain and undergo cooperative denaturation. We have demonstrated that a protein with a single structural domain, binase, and a protein with multiple structural domains, porcine pepsin, contain fewer cooperative regions (energetic domains) under the conditions optimal for their functional activity. The study was performed by combining a CD analysis of the structural changes in the proteins during thermal denaturation and under various solvent conditions with thermodynamic properties observed by scanning microcalorimetry. Estimates of secondary structure were obtained from CD spectra, taking side-chain CD into account. It was found that neither of the proteins show any changes in secondary structure or local environment of aromatic amino acids upon separation of the energetic domains. The structural regions in binase corresponding to energetic domains were identified. It was shown that binase is converted from a single cooperative system into two separate energetic domains when ion pairs are disrupted, whereas the size of cooperative units in pepsin decrease as the electrostatic repulsion between regions in the molecule increases.
Open Access
Mechanisms of RNA polymerase II-mediated transcription
(Colorado State University. Libraries, 2007) Fletcher, Aaron Glenn Louis, author; Stargell, Laurie, advisor
Transcription by RNA Polymerase II (RNAPII) is a critical step in controlling biological events such as cell growth, cell differentiation, response to environmental change, homeostasis, and disease. The regulation of transcription initiation of some genes is controlled at the level of TBP and RNAPII recruitment to the promoter. At such genes, the binding of TBP/RNAPII is the rate-limiting step for gene expression. Other genes already have TBP/RNAPII occupying the promoter before induction of gene expression, and the rate limiting step is no longer recruitment of TBP/RNAPII. These genes are collectively known as post-recruitment regulated genes. The yeast CYC1 gene is a post-recruitment regulated gene and serves as an excellent model for understanding the mechanism behind post-recruitment regulation. A TBP recruitment bypass screen was developed to investigate the mechanism of post-recruitment regulation. The results of the bypass screen revealed that SAGA and Mediator play important roles in post-recruitment regulation. Further analysis of SAGA uncovered a new function: that suggests SAGA is important in recruitment of Mediator to post-recruitment regulated genes. In addition to RNAPII and TBP, the CYC1 gene was found to have TFIIH, capping enzyme and serine 5 phosphorylation of the RNAPII C-terminal domain occupying the promoter in the uninduced condition. These results indicate that much of the Pre-Initiation Complex (PIC) occupies the CYC1 promoter in the uninduced state. In addition to PIC occupancy at CYC1, a conserved and essential protein, Spn1, is found to occupy the promoter during uninduced conditions. To further understand the role of this essential protein, genome localization studies and transcription profiling were performed. These studies suggest that in addition to playing an important role in post-recruitment regulation of gene expression, Spn1 may be involved in the transcription of ribosomal proteins. Taken together, this body of work contributes significantly to understanding the regulatory mechanisms of post-recruitment regulation.
Open Access
Structural and functional studies on the chromatin and nucleosome binding proteins
(Colorado State University. Libraries, 2007) Chodaparambil, Jayanth Velandy, author; Luger, Karolin, advisor
The approximately two meters of eukaryotic DNA are compacted within the confines of the nucleus by hierarchical packing with an equal amount of histone proteins to form chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. Highly compacted DNA is very accessible to the transcription machinery. To understand the mystery behind the two opposing functions of the chromatin, it is essential for us to study nucleosome and chromatin structure in detail.
Open Access
The influence of histone orthologues, histone variants and post-translational modifications on the structure and function of chromatin
(Colorado State University. Libraries, 2008) Resch, Michael George, author; Hansen, Jeffrey C., advisor; Luger, Karolin, advisor
Two meters of DNA is packaged into the nucleus of each eukaryotic cell in the form of chromatin. DNA wraps around a protein histone octamer to form a nucleosome, the fundamental repeating unit of chromatin. The highly basic histone octamer contains two copies each of H2A, H2B, H3 and H4 to form the protein core of the nucleosome. There is a dynamic interplay of accessibility which compacts DNA yet allows access for fundamental cellular processes like transcription and DNA replication. This thesis investigates how histone variants and post-translational modifications contribute to the level of chromatin compaction. I demonstrated that defined nucleosomal arrays made with histones from multiple species oligomerize at different concentrations of MgCl2. A comparison of endogenous and recombinant Drosophila melanogaster histone octamers showed that this is unlikely due to posttranslational histone modifications, but likely a result of subtle changes in the sequences constituting the histone tails and structured surface of the histone octamer. I investigated the effect of incorporation of the centromere specific H3 histone variant centromere protein - A (CENP-A) into nucleosomes and nucleosomal arrays. Despite the fact that CENP-A shares only 60% sequence homology within the structured domain of major-type H3 (15% in the N-terminal domain), CENP-A (together with the other three core histones) forms nucleosomes and condensed nucleosomal arrays comparable to major-type H3. Post-translational modifications (PTM) contribute to the regulation of chromatin structure. I have analyzed the effect of H3 lysine 56 acetylation on nucleosome structure and chromatin condensation. This modification was previously thought to disrupt nucleosome structure. I developed methods to enzymatically acetylate large amounts of H3 specifically at Lys 56, and demonstrated that histone octamers containing H3-K56Ac form canonical nucleosomes. However, nucleosomal array condensation is compromised by this particular PTM. Together, these studies suggest that even subtle variations in histone sequence or post-translational modifications result in differences in chromatin higher order structure.
Open Access
New functions of the SAGA complex in regulation of transcription by RNA polymerase II
(Colorado State University. Libraries, 2008) Chen, Xu, author; Stargell, Laurie, advisor
The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex plays a role in Gal4-mediated transcriptional activation via delivery of TATA-binding protein (TBP) to Gal4-responsive promoters. Little is known about the impact of the sequence of the TATA element in the core promoter in this process. To investigate the SAGA complex regulatory function at different TATA element sequences, we compared a consensus element (TATA) to an off-consensus element (CATA) in the kinetics of Gal4-dependent gene activation, PIC occupancy, the requirement of SAGA components, and the histone acetylation state. We have found a new function of SAGA carried by subunits Gcn5, Ada2 and Spt8: TATA-element-censoring. This function enhances transcription driven by the consensus TATA element and represses transcription driven by off-consensus TATA elements. This functions works at both synthetic promoters and the endogenous GAL promoters. Via a genetic screen, Swi/Snf and RSC complexes were also identified with TATA-censoring function. Our study suggests that the new function involves TBP delivery, histone acetylation and histone eviction.
Open Access
Tax deregulation of host-cell proteins
(Colorado State University. Libraries, 2008) Kim, Young-Mi, author; Nyborg, Jennifer K., advisor
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked to an aggressive and generally fatal malignancy called adult T-cell leukemia (ATL) and to a chronic inflammatory neurological disease. Only a small percentage of infected individuals develop ATL following a prolonged latency period of up to 30 years post infection. The dominant mechanism of virus transmission in an infected individual is through clonal expansion of HTLV-1 infected cells. The HTLV-1-encoded protein Tax is the prominent player in promoting mitotic replication. Tax is also directly linked to malignant transformation and the etiology of ATL. Tax is a potent transcriptional activator that stimulates HTLV-1 viral gene expression. Three 21 base pair repeat enhancer elements called viral cyclic AMP response elements (vCREs), located in the HTLV-1 transcriptional control region, are critical to Tax-activated transcription. Tax associates with the vCREs through protein-DNA interactions and through protein-protein interaction with the cellular transcription factor cAMP response element binding (CREB) protein. Together this complex recruits the cellular coactivators CBP/p300. The role of Ser133 phosphorylated CREB in mediating Tax function in HTLV-1 transcription has long been controversial. Our data reveal that CREB phosphorylation is absolutely required for viral Tax transactivation. Consistent with this, Tax induces constitutively elevated levels of phosphorylated CREB in vivo and in vitro. We further investigated the mechanism of Tax-mediated CREB phosphorylation and uncovered a novel function of Tax: stimulation of CREB phosphorylation via the Ca2+/Calmodulin (CaM)-dependent protein kinase (CaM kinase) pathway to promote viral transcription. In addition to Tax-dependent CREB phosphorylation, we found that Tax upregulates B-cell lymphatic leukemia protein 3 (Bcl-3) and cyclin D1 expression, two key determinants of cell fate. Furthermore, Tax interacts with Bcl-3 in vivo and in vitro. Deregulation of these key host-cell proteins by Tax may contribute to the transformation of T-cells.
Open Access
Role of polyglutamylation in nucleosome assembly protein 1 (NAP1) function
(Colorado State University. Libraries, 2008) Subramanian, Vidya, author; Luger, Karolin, advisor
The organization of DNA into chromatin requires the systematic deposition of the histones onto the DNA template. Chromatin function requires the dynamic exchange of the histone components during replication and transcription. Deposition and exchange is mediated in part by a family of proteins generally referred to as histone chaperones. It has been shown recently that recombinant yeast NAP1 (yNAP1) is capable of promoting ATP-independent histone exchange and nucleosome sliding in vitro, and this ability is specifically attributed to the highly acidic C terminal tail of the protein. Drosophila NAP1 (dNAP1) has a shorter acidic C terminus than yNAP1. Preliminary data in the lab suggests that recombinant wild-type dNAP1 is incapable of this nucleosome dissociation. Native dNAP1 purified from Drosophila embryos, on the other hand, is capable of nucleosome dissociation. In this study we reveal the presence of a unique post-translational modification, polyglutamylation in native dNAP1, which restores the nucleosome dissociation function to recombinant dNAP1. We have also been able to identify two target sequences, as well as the number of glutamyl units associated with these modifications using mass spectrometric analysis (MALDI & MS/MS). The modification at the CTAD (C-terminal acidic tail domain) could compensate for the lesser amount of acidic amino acid in dNAP1 and may account for the gain in nucleosome dissociation function. The second polyglutamylation site is located at the NLS (Nuclear Localization Sequence) (based on the conserved core domain of yNAP1 and dNAP1).
Open Access
Insulin and IGF-I prevent brain atrophy in diabetic rats independently of hyperglycemia
(Colorado State University. Libraries, 2009) Šerbedžija, Predrag, author; Ishii, Douglas N., advisor
The causation of brain atrophy associated with dementia in diseases such as diabetes and Alzheimer's Disease (AD) is very poorly understood. There are 20.8 million patients in US with diabetes, 4.5 million with AD, and the incidence of both is rising. Their combined annual treatment cost (direct and indirect) for patients with dementia exceed $148 billion. Reduced insulin and insulin-like growth factor (IGF) signaling is a common biochemical feature in brains of diabetic as well as AD patients. Rodents with knockout of brain neuronal insulin receptor display no change in glucose utilization, neurodegeneration, nor impaired learning/memory. However, IGFs may substitute for insulin or be required for its activity. The literature is virtually silent concerning the role of the widespread insulin receptors in the brain. It is possible that brain atrophy is the consequence of a concomitant decline in IGF as well as insulin levels. The causation of adult brain atrophy is understudied, and identification of the factors that help maintain normal brain mass may provide hints as to the causation of neurodegenerative disorders.
Open Access
Molecular basis of yeast prion formation
(Colorado State University. Libraries, 2009) Toombs, James A., author; Ross, Eric, advisor
Amyloid fibers are highly organized protein aggregates that are associated with many fatal diseases. Prions represent a unique class of amyloid fibers that are distinguished by their infectivity and inheritability. In the yeast S. cerevisiae, there are several known prion forming proteins. Since the discovery of the first yeast prions in the early 1990s, they have provided a useful model system for studying the biology of prion proteins. While it has been determined that amino acid composition is important to prion formation, there has not yet been any quantitative study aimed at determining how composition promotes or inhibits prion formation. Without this knowledge, our understanding of the events that drive prion formation and our ability to identify new prion-forming proteins is severely limited. In this dissertation, we describe our experiments with the yeast prion protein Sup35p that have illuminated the sequence requirements for yeast prion formation. From these results, we conclude that: (i) amino acid composition, not primary sequence, is the major driving force behind yeast prion propagation, and (ii) prion formation occurs in domains characterized by relatively few prion promoting residues dispersed throughout an intrinsically disordered region.
Open Access
Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex
(Colorado State University. Libraries, 2009) Yang, Chenghua, author; Luger, Karolin, advisor
Methyl-CpG Binding Protein (MeCP2) is an abundant chromatin associated protein that is important in maintaining human health; mutations in this protein cause Rett Syndrome, a neurodevelopmental disease that is a common cause of mental retardation and autism in females. MeCP2 was initially identified as a protein that recognizes the genetic DNA methyl-CpG mark and it was thought to repress gene transcription by recruiting histone deacetylases. Recent studies show that MeCP2 can both repress and activate gene transcription. It also binds chromatin in the absence of the methylation mark, suggesting that its mode of action is more complex than previously assumed. The observation that MeCP2 compacts nucleosomal arrays in vitro and mediates silent chromatin loop formation in vivo suggests a novel mechanism by which MeCP2 regulates gene expression. To further characterize the interplay between MeCP2 and chromatin, it is important to understand the interactions between MeCP2 and nucleosomes, the fundamental component of chromatin. We used biochemical and biophysical approaches to study the interplay between MeCP2 and nucleosomes. Gel mobility assays showed that although MeCP2 can interact with a nucleosome with or without extra nucleosomal DNA, it has a higher affinity for nucleosomes with extra nucleosomal DNA. The N-terminal portion of human MeCP2 (amino acids 78-305) is sufficient to establish this interaction. Size-exclusion chromatography combined with multi-angle light scattering and fluoresecence resonance energy transfer (FRET) assays demonstrated that this interaction occurs at a 1:1 molar ratio and that MeCP2 brings the extra nucleosomal DNA ends in a closer proximity. Small angle X-ray scattering (SAXS) revealed the formation of a more compact complex when MeCP2 interacts with nucleosome with (versus without) extra nucleosomal DNA, indicating that the extra nucleosomal DNA is important in organizing the MeCP2-nucleosome complex. Our data suggest a model in which MeCP2 compacts chromatin by changing the extra nucleosomal DNA path. X-ray crystallography is also used to characterize the nucleosome-MeCP2 complex. Crystals of the nucleosomes with extra nucleosomal DNA in complex with MeCP2 were obtained and diffracted to 5.2 Å. Although MeCP2 dissociated from the crystals after soaking in cryo-protectant, the electron density map reveals the path of extra nucleosomal DNA which may be organized by MeCP2.
Open Access
The amyloid beta dimer/trimer: a potent stimulator of neuronal amyloid beta secretion and cofilin-actin rod formation
(Colorado State University. Libraries, 2010) Marsden, Ian Thomas, author; Bamburg, James R., advisor; Di Pietro, Santiago M., committee member; Partin, Kathryn M., committee member
Amyloid beta (Aβ) peptides, a heterogeneous mixture of 39-43 amino acid peptides produced from β- and γ-secretase cleavage of the amyloid precursor protein (APP), are one of the causative agents of Alzheimer disease (AD). Although sensitive enzyme-linked immunosorbent assays (ELISAs) for specific rodent Aβ peptides and for total and specific human Aβ peptides have been commercially available, no commercial assay for total rodent Aβ was available when we began these studies. Such an assay is desirable to determine the effects of the human Aβ peptides on production of Aβ from cultured rodent neurons, the major model system used in AD research. Here we report an ELISA for total rodent Aβ and show that it can be used without interference from physiologically relevant concentrations of human Aβ. We then apply the assay to measure the production of Aβ in cultured dissociated rat cortical neurons and rat and mouse hippocampal organotypic slices in response to oxidative stress or treatment with human Aβ dimer/trimer (Aβd/t) obtained from culture medium of Chinese hamster ovary cell line 7PA2 expressing a mutant form of human amyloid precursor protein. Neither of the treatments leads to accumulation of intracellular Aβ peptides. Peroxide increases Aβ secretion by about 2 fold, similar to results from previous reports that used an immunoprecipitation and western blot assay. Of greater significance is that physiologically relevant concentrations (250 pM) of human Aβd/t increase rodent Aβ secretion by >3 fold over 4 days, providing support for an Aβ-mediated feed-forward model of AD progression. The over two fold increase in rodent Aβ secreted in response to human Aβd/t was nearly identical between organotypic hippocampal slices of TAU knock-out mice and TAU knock-out mice expressing the human tau transgene, demonstrating that tau plays no role in the enhanced production of Aβ. Previous studies showed oligomers of synthetic amyloid beta (Aβ1-42) induced cofilin activation and formation of cofilin-actin rods in a neuronal subpopulation of rat hippocampus primarily localized within the dentate gyrus. Here we demonstrate that Aβd/t at ~250 pM is more potent in rod induction in both dissociated hippocampal neuronal cultures and organotypic slices than is 1 μM synthetic Aβ as typically prepared oligomers, about a 4000 fold difference. Treatment of the Aβd/t fraction with an Aβ-neutralizing antibody eliminates its rod inducing activity. Traditionally prepared synthetic Aβ oligomers contain SDS-stable trimers and tetramers, but are devoid of dimers. When synthetic human Aβ was incubated under conditions that generate a tyrosine oxidized dimer, the concentration that was required to induce rods decreased dramatically. The oxidized dimer had a maximum rod-inducing activity at ~2 nM (10 ng/mL), suggesting it is the presence of the SDS-stable tyrosine oxidized Aβ dimer in a low-n state that is largely responsible for the potency of the secreted Aβd/t. Aβd/t-induced rods are highly localized to the dentate gyrus and mossy fiber pathway and form more rapidly (significant over controls by 2 h compared to 8 h for those induced by synthetic Aβ-oligomers). Aβd/t-induced rods are reversible, disappearing by 24 h after washout. Cofilin dephosphorylation in response to Aβd/t is greatest within the hippocampal regions of rod formation. Overexpression of cofilin phosphatases slingshot and chronophin increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t treatment both in dissociated neurons and organotypic slice cultures. Overexpression of the cofilin kinase, LIM kinase 1, inhibits Aβd/t-induced rod formation. Together these data support a mechanism through which Aβd/t produces selective synaptic dysfunction affecting learning and memory at least in part via primary effects on cofilin regulation and rod formation in sensitive hippocampal regions.
Open Access
Activation of gene expression in yeast
(Colorado State University. Libraries, 2010) Lee, Sarah Kathleen, author; Stargell, Laurie Ann, 1963-, advisor; Luger, Karolin, committee member; Nerger, Janice Lee, 1960-, committee member; Nyborg, Jennifer Kay, committee member; Paule, Marvin R., 1943-, committee member; Ross, Eric D., committee member
Transcription is the generation of RNA from the DNA template, and is the fundamental aspect of gene expression. As such, the initiation of transcription at genes that are transcribed by RNA polymerase II (RNAPII) is a major control point in gene expression. Organisms across the evolutionary spectrum possess genes whose transcription is regulated after recruitment of RNAPII to the promoter, or postrecruitment. This regulatory strategy has been observed in bacteria, yeast, worms, flies, and humans. Therefore, postrecruitment regulation is a conserved strategy for controlling gene expression. Genome-wide studies in Drosophila and humans demonstrate that a significant portion of these genomes are postrecruitment regulated. Recent studies in humans indicate two biologically important activators (p53 and c-myc) are involved in releasing paused polymerases from promoter DNA1,2. These regulators of cell growth and differentiation are both implicated in carcinogenesis. Thus, further understanding how activators regulate the transition from an inactive to active polymerase will prove crucial in our understanding of transcriptional regulation and human diseases. Coactivators are conserved, multiprotein complexes involved in regulating the transcription process at most genes. Yet, virtually nothing is known about the role of coactivators at postrecruitment regulated genes in yeast. The work presented in this dissertation details the identification of postrecruitment functions of two coactivators, the Mediator and SAGA complexes. My studies reveal that coactivators act as intermediaries with activator proteins to stimulate transcription after the recruitment of RNAPII to the promoter. Further, this work demonstrates that this conserved class of factors plays a role in postrecruitment regulation, a previously unappreciated aspect of coactivator function. Analysis of Mediator function at the postrecruitment regulated CYC1 gene revealed a functional submodule of the Mediator complex that is required for triggering the preloaded polymerase at the CYC1 promoter into an active polymerase. This requirement exists even when two different activator proteins control CYC1 expression, Hap2/3/4/5 and Yap1. Strikingly, this submodule is not required for activation of a recruitment regulated Yap1-dependent gene, GTT2. The Yap1 activator controls the expression of a number of genes during oxidative stress in yeast. Oxidative stress is a damaging condition that haunts all aerobic organisms, and is linked to many human ailments. Yeast respond to this biological assault with a rapid activation of many genes. My investigation of Yap1-dependent transcription demonstrated that postrecruitment regulation is more prevalent in yeast than previously thought. Analysis of SAGA function at Yap1-dependent genes revealed that Yap1 utilizes SAGA during oxidative stress. Despite a common reliance on the SAGA coactivator for expression, each gene has different specific SAGA requirements. This demonstrates an important role for the SAGA coactivator during the important biological response to oxidative stress, and the complexity inherent in transcriptional regulation. In sum, my findings illustrate the mechanisms of activated transcription yeast utilize in response to important biological stimuli. This work significantly advances our understanding of the regulation of transcription after RNAPII arrives at the promoter. It also reveals the novel role that coactivators play in stimulating transcription at the group of genes that are regulated in this fashion.
Open Access
DENVax live attenuated chimeric dengue vaccine
(Colorado State University. Libraries, 2010) Mulhern, Kaitlyn, author; Nyborg, Jennifer, advisor; Livengood, Jill, advisor; Stinchcomb, Dan, committee member; Blair, Carol, committee member
Dengue viruses are endemic in regions inhabited by roughly one half of the world’s population and can cause symptoms ranging from a headache and fever to even death. There is no vaccine to prevent infection by dengue viruses and the only current protection and prevention is mosquito control. The dengue viruses and the Aedes aegypti mosquito that carries them are spreading to more areas of the world and are therefore posing an even greater threat to public health. There is an urgent need for an effective vaccine that confers protection against infection by all four dengue serotypes. Several companies are currently developing dengue vaccines using different technologies including a live attenuated virus vaccine, DNA vaccine, and a chimeric vaccine in the yellow fever virus vaccine backbone. Inviragen, Inc., a biotech company, in collaboration with the Center for Disease Control and Prevention (CDC), is developing a chimeric live attenuated virus vaccine that is currently in clinical testing. The Inviragen/CDC dengue vaccine is based on an attenuated dengue-2 virus, called PDK-53, which was developed by passaging the virus 53 times in primary dog kidney cells (PDK). The attenuating mutations in the dengue-2 PDK-53, which are in the nonstructural genes of the virus, are well defined and characterized. This is essential in a live attenuated vaccine in order to monitor the stability of the mutations throughout vaccine manufacture and development. The four separate serotype-specific vaccine viruses are constructed by inserting the appropriate structural E and prM gene sequences into the nonstructural genes of the attenuated dengue-2 PDK-53 backbone. The phenotypic properties including plaque size, temperature sensitivity, peak titers, efficiency of replication, and neurovirulence, showed that the ehimeric viruses for each serotype were attenuated in comparison to the wild type parental viruses. Tetravalent formulations with the chimeric D2/1, D2/3 and D2/4 viruses as well as the D2-PDK53 virus were tested for antibody responses in AG 129 mice. Two separate projects involving Inviragen’s dengue vaccine, DENVax, are discussed in this thesis. In the first project, assays were developed to purify and quantify the residual host cell DNA in the vaccine using real time qPCR. Initially, the purification of host cell DNA present in research grade vaccine lots was optimized and internal controls were generated to monitor the efficiency of DNA extraction from the samples. The purified DNA was then amplified by PCR and quantified using two fluorescence-based methodologies, SYBR Green and TaqMan. The SYBR Green method was used to test the virus samples and reproducible results were obtained. The TaqMan assay allows for multiplexing, which allows simultaneous detection of two PCR products; Vero cell DNA present in virus samples and an internal control. The multiplexing TaqMan reactions need to be further optimized. In the second project, three separate “second generation” DENVax4 viruses were designed and generated. Of the three, two viable infectious viruses were generated and tested for phenotypic growth in vitro, and sequenced. These viruses grew to similar titers and phenotypes to the existing DENYax4, and immunogenicity testing in AG 129 mice is planned.
Open Access
The influence of H2AX and γH2AX on chromatin condensation
(Colorado State University. Libraries, 2010) Norskog, Sarah Samaya, author; Hansen, Jeffrey, advisor; Luger, Karolin, committee member; Zabel, Mark, committee member
Chromatin composition and structure are essential for the condensation of the genome and the regulation of a wide range of cellular activities. Chromatin condensation is thought to be controlled predominantly through interactions mediated by the unstructured amino terminal domains of the core histones H4, H3, H2A and H2B. In addition to the amino terminal domain, histone H2A contains an unstructured carboxyl terminal domain. Multiple H2A variants, many differing from major type H2A in this C-terminal domain sequence, have been identified. The most studied of variant is H2AX, which contains a conserved serine residue that becomes phosphorylated following double strand DNA breakage (yH2AX). Although the phosphorylation of the H2AX has been identified as a key step in major genomic activities, the basic mechanism by which it functions remains controversial. Here, I have determined the structural role of H2AX and yH2AX using in vitro assays which utilize defined nucleosomal arrays. H2AX and yH2AX alter chromatin folding under high salt concentrations but show no discernable differences in low concentrations of salt or under conditions which favor oligomerization. The phosphorylation of H2AX does not alter the folding or oligomerization relative to the unphosphorylated form, indicating yH2AX more likely functions as a signaling and recruitment motif rather than as a chromatin secondary structure remodeling factor.
Open Access
Biophysical, structural, and functional studies of histone binding proteins
(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
Open Access
Molecular characterization of the protein-protein interaction between HTLV-1 Tax and GSK-3ß
(Colorado State University. Libraries, 2010) Wang, Guoliang, author; Nyborg, Jennifer, advisor; Bamburg, James R., committee member; Quackenbush, Sandra L., committee member; Laybourn, Paul J., committee member
The human T-cell leukemia virus type 1 (HTLV-1) encodes a viral oncoprotein termed Tax, which plays a major role in transforming HTLV-1- infected cells. Tax is a potent transcriptional activator that stimulates HTLV-1 viral and cellular gene transcription. In addition, Tax disrupts a number of cell signaling pathways involved in cell growth and survival. Glycogen synthase kinase-33 (GSK-33) is a ubiquitously expressed serine/threonine kinase present in all eukaryotic cells, which functions as a critical regulator of a wide range of cell signaling pathways. As GSK-33 is constitutively active in resting cells, it is primarily regulated through inhibition. Ser-9 phosphorylation is inhibitory to the kinase activity of GSK-33. Deregulation of GSK-33 has been linked to many human diseases such as Alzheimer’s disease and cancers. It has been reported in Aida Ulloa’s thesis that Tax inhibits GSK-33 kinase activity toward both primed and non-primed substrates through direct association. To delineate the protein-protein interaction between Tax and GSK-33, we compared the amino acid sequence of Tax with a well-characterized short peptide deriving from the GSK-33 interacting domain (GID) of Axin, and found that Tax contains a notable amino acid sequence homology to Axin GID. The region spanning Tax amino acids 185 - 205 has 24% sequence identity and 19% similarity with Axin GID. We named this region the putative Tax GID. We characterized the putative Tax GID biochemically, and discovered that a longer peptide (Tax aa. 138 - 205) of the putative Tax GID strongly inhibits GSK-3(3 kinase activity in vitro. Bioinformatics computation was used to predict the secondary structure of the Tax GID, which was further used in our docking test to identify a potential binding interface in GSK-3p. This was tested by GST pulldown and Co-IP assays using point and deletion mutants. In addition, the effects of Tax-GSK-3(3 interaction on the downstream (3-catenin and NFAT pathways were characterized by luciferase reporter assays. However, unexpectedly, we observed that Tax expression has little effects on p-catenin and NFAT transcriptional activation.
Open Access
Mutational analysis of the human histone chaperone, Nap1, in nucleosome disassembly at the HTLV-1 promoter
(Colorado State University. Libraries, 2010) Kolean, Jennica Laura, author; Nyborg, Jennifer K., advisor; Stargell, Laurie A., committee member; Bailey, Susan M., committee member
The human genome is packaged to fit within the confines of the nucleus through the interaction with four core histone proteins, H2A, H2B, H3 and H4. These proteins organize the genetic material, however they also make it difficult for the cells to access the information stored within the DNA sequence for processes such as transcription and replication. One of the mechanisms by which the genetic information can be accessed is post-translational modifications of the histone tails. Post-translational modifications, such as acetylation, act to neutralize charges on the histone tails and also serve to create new binding sites for other proteins. These modifications have been associated with decompaction of condensed chromatin, alteration of nucleosome structure, and partial or complete disassembly of the histone octamer. Our laboratory uses human T-cell leukemia virus, type 1 (HTLV-1) as a model for studying eukaryotic transcription activation and gene regulation. Previous studies using chromatin immunoprecipitation to look at the HTLV-1 promoter have correlated transcription activation with a decrease in post-translational modifications that are traditionally associated with gene activation. This decrease in activating modifications was due to a decrease in histone occupancy at the promoter in vivo. To recapitulate the results observed in vivo, we developed an immobilized template assay using the biotin labeled HTLV-1 promoter fragment bound to a magnetic streptavidin coupled bead. Nucleosome disassembly at the HTLV-1 promoter is dependent on the presence of the virally encoded Tax protein, as well as the phosphorylated form of the cellular protein cyclic-AMP response element binding protein (pCREB), cellular coactivators CREB binding protein (CBP)/p300, acetyl coenzyme A (acetyl CoA), acceptor DNA and nucleosome assembly protein (Nap1). Tax and pCREB recruit the histone acetyltransferase, CBP/p300, which acetylates histone tails prior to disassembly of the octamer. Nap1 is unique in this reaction because this was the first example of a histone chaperone supporting disassembly of the entire octamer in an acetyl CoA dependent fashion, independent of ATP consumption or the presence of chromatin remodeling complexes. In this study we examined the domains of Nap1 required for in vitro nucleosome disassembly at the HTLV-1 promoter template through a series of rationally designed deletion mutants. Crystal structures of yeast Nap1 and SET/TAF-Iβ; were used as models for designing mutations in the human Nap1 protein. Our results show that the minimal domain of human Nap1 able to support nucleosome disassembly is contained within amino acid residues 196-290. Using histone binding assays, we also found that the ability to disassemble nucleosomes is independent of histone interaction in vitro. Removal of the β-hairpin that is required for Nap1 oligomerization renders the protein unable to support disassembly. This suggests that the oligomeric form of Nap1 is required for nucleosome disassembly at the HTLV-1 promoter.
Open Access
Solving the crystallographic structure of the Cl2J construct and occupancy titration trials to quantitatively determine isomer ratio
(Colorado State University. Libraries, 2011) Rummel, Brittany L., author; Ho, P. Shing, advisor; Curthoys, Norman, committee member; Bamburg, James, committee member; Kennan, Alan, committee member
Halogen atoms are commonly found in biological organic compounds such as plastic polymers, flame retardants, coolant fluids, insecticides and herbicides. Halogens are known to mediate neurotransmitters in the brain and are required for the production of many hormones (i.e. thyroxine). Because halogen atoms are frequently incorporated in pharmaceuticals and antibiotics (i.e. clindamycin and chloramphenicol), it is important to characterize the interactions that those atoms participate in. Currently, there is little information known about halogen bonds and these interactions are not modeled accurately by molecular simulations. The long-term objective of Dr. Shing Ho's laboratory has been to characterize halogen bonds through structural and energetic determinations. As part of that larger goal, the studies in this thesis aim to address the structure-energy relationship of chlorinated halogen bonds or X-bonds. The experimental assay that allowed the study of halogen bonds is the 4-stranded DNA Holliday junction. Incorporating engineered halogen bonds into the structure results in halogen bonds competing energetically against hydrogen bonds for stabilization of the junction. The structure that was refined in order to analyze chlorinated halogen bonds is referred to as the Cl2J construct. The Cl2J construct is a crystallized Holliday junction crystal in which 2 chlorine atoms are incorporated into the structure as chlorinated uracil nucleotides, and thus, sets chlorine halogen bonding energies and hydrogen bonding energies in opposition. Occupancy titrations were conducted to quantify isomeric ratios of halogen bond versus hydrogen bond stabilized junctions (X- and H-isomers, respectively) within these crystals. The initial estimate of the isomer ratios of the Cl2J construct was 50/50 X-to-H-isomer from the initial electron density maps. The crystallographic model and subsequent occupancy titration trials actually indicate a higher ratio of approximately 3/1 X-to-H-isomer ratio, respectively. The occupancy titrations and crystallographic models of other constructs, F2J, Br2J and I2J, were analyzed in comparison to the Cl2J construct in order to define a protocol that accurately quantifies these isomeric ratios. Differential scanning calorimetry (DSC) studies are also presented to corroborate in solution any conclusions drawn from occupancy titrations in the crystals.
Open Access
Role of basic and hydrophobic residues in the poliovirus polymerase elongation complex and the structure of a coxsackievirus polymerase elongation complex
(Colorado State University. Libraries, 2011) Kortus, Matt, author; Peersen, Olve, advisor; Ho, P. Shing, committee member; Suchman, Erica, committee member
Picornaviruses encode for and require a viral RNA-dependent RNA polymerase (RdRP) for genome replication. This enzyme synthesizes negative-sense RNA from the infecting positive sense genome producing a replicative intermediate. The negative sense RNA then serves as a template for synthesis of additional positive-sense RNA. To efficiently replicate the genome, RdRPs must form a stable and processive elongation complex (EC) by binding RNA, incorporating the first templating nucleotide, and undergoing a necessary conformational. Upon completion of these steps that comprise initiation, the newly formed EC is capable of rapidly replicating the viral genome. The work presented in this thesis 1) investigates the role that several basic and hydrophobic residues serve in forming and maintaining the poliovirus (PV) EC and 2) presents the crystal structure of a coxsackievirus (CV) EC. To determine the role of that several arginines, lysines, and tyrosines play in the PV polymerase, we assessed whether mutations to these residues affect initiation, elongation, or stability of the EC. The data indicates the basic residues within the fingers domain of the PV polymerase have a major role in binding RNA. In addition, data shows two tyrosine residues in particular are critical for formation and maintenance of the EC. Overall, the data provides evidence the fingers domain interacts with the template RNA in a manner not captured by crystal structures. Finally, we have solved the structure of a CVEC stalled after incorporation of four nucleotides. The CVEC structure closely matches the previously solved PVEC structure. In addition, one crystal form produced an elongation complex trapped in a translocation intermediate state.
Open Access
Nucleosomal array condensation: new insights into an old "tail"
(Colorado State University. Libraries, 2011) Sorensen, Troy C., author; Hansen, Jeffrey C., advisor; Stargell, Laurie, committee member; Luger, Karolin, committee member; Bailey, Susan, committee member
The DNA present within the nucleus of each human somatic cell, when extended end to end, would span a distance of about one meter. The first level of compaction critical to fitting the entire genome into the nucleus is the nucleosome, consisting of 147 base pairs of DNA wrapped 1.7 times around an octameric structure composed of the four core histones H2A, H2B, H3 and H4. Nucleosomes separated by up to 80 base pairs of linker DNA called nucleosomal arrays compact the DNA further through short range intra-array and long range inter-array contacts that generate different levels of higher order condensed structures. This dissertation investigates the involvement of the core histone "tail" domains as well as the influence of the H3 centromeric variant CENP-A in nucleosomal array condensation events. In vitro, 12-mer nucleosomal arrays condense intra- and inter-molecularly through nucleosome-nucleosome interactions driven primarily by the core histone tail. This dissertation details the contributions and the molecular determinants of the histone tail domains to the condensation processes. Importantly, we found that the H3 and H4 tail domains were the largest contributors to array condensation. The mode of action used by the H4 tail domain in intra- and intermolecular condensation centered on the following determinants: 1) position of the H4 tail, 2) amino acid composition, 3) positive charge density and 4) tail domain length. Importantly, the primary sequence of the H4 tail was found to not be an important molecular determinant. To date no study has been performed to determine short-range compaction between "bulk" H3 containing and H3 centromeric specific variant, CENP-A chromatin. 12-mer nucleosomal arrays containing either H3 or CENP-A histones were reconstituted and tested for their ability to fold intra-molecularly. Major finding include that CENP-A containing nucleosomal arrays assemble in the same stepwise manner as conical arrays and were always more compact than H3 containing arrays at every salt concentration tested. The increased compaction was found to be in part due to a lysine to arginine mutation at position 49 of CENP-A.