Matching Items (21)
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- All Subjects: Biochemistry
- Creators: LaBaer, Joshua
- Member of: Theses and Dissertations
Description
ABSTRACT
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
ContributorsMorales Betanzos, Carlos (Author) / LaBaer, Joshua (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology.
ContributorsFlores, Julia Anne (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2012
Description
Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine.
ContributorsDunn, Matthew Ryan (Author) / Chaput, John C (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2015
Description
Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions.
ContributorsPetritis, Brianne Ogata (Author) / LaBaer, Joshua (Thesis advisor) / Lake, Douglas (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
The influenza virus, also known as "the flu", is an infectious disease that has constantly affected the health of humanity. There is currently no known cure for Influenza. The Center for Innovations in Medicine at the Biodesign Institute located on campus at Arizona State University has been developing synbodies as a possible Influenza therapeutic. Specifically, at CIM, we have attempted to design these initial synbodies to target the entire Influenza virus and preliminary data leads us to believe that these synbodies target Nucleoprotein (NP). Given that the synbody targets NP, the penetration of cells via synbody should also occur. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. The focus of my honors thesis is to explore how synthetic antibodies can potentially inhibit replication of the Influenza (H1N1) A/Puerto Rico/8/34 strain so that a therapeutic can be developed. A high affinity synbody for Influenza can be utilized to test for inhibition of Influenza as shown by preliminary data. The 5-5-3819 synthetic antibody's internalization in live cells was visualized with Madin-Darby Kidney Cells under a Confocal Microscope. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. Expression of NP over 8 hours time was analyzed via Western Blot Analysis, which showed NP accumulation was retarded in synbody treated cells. The data obtained from my honors thesis and preliminary data provided suggest that the synthetic antibody penetrates live cells and targets NP. The results of my thesis presents valuable information that can be utilized by other researchers so that future experiments can be performed, eventually leading to the creation of a more effective therapeutic for influenza.
ContributorsHayden, Joel James (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics.
ContributorsWoolley, Christine F (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2015
Description
Currently, treatment for multiple myeloma (MM), a hematological cancer, is limited to post-symptomatic chemotherapy combined with other pharmaceuticals and steroids. Even so, the immuno-depressing cancer can continue to proliferate, leading to a median survival period of two to five years. B cells in the bone marrow are responsible for generating antigen-specific antibodies, but in MM the B cells express mutated, non-specific monoclonal antibodies. Therefore, it is hypothesized that antibody-based assay and therapy may be feasible for detecting and treating the disease. In this project, 330k peptide microarrays were used to ascertain the binding affinity of sera antibodies for MM patients with random sequence peptides; these results were then contrasted with normal donor assays to determine the "immunosignatures" for MM. From this data, high-binding peptides with target-specificity (high fluorescent intensity for one patient, low in all other patients and normal donors) were selected for two MM patients. These peptides were narrowed down to two lists of five (10 total peptides) to analyze in a synthetic antibody study. The rationale behind this originates from the idea that antibodies present specific binding sites on either of their branches, thus relating high binding peptides from the arrays to potential binding targets of the monoclonal antibodies. Furthermore, these peptides may be synthesized on a synthetic antibody scaffold with the potential to induce targeted delivery of radioactive or chemotherapeutic molecular tags to only myelomic B cells. If successful, this would provide a novel alternative to current treatments that is less invasive, has fewer side effects, more specifically targets the cause of MM, and reliably diagnoses the cancer in the presymptomatic stage.
ContributorsBerry, Jameson (Co-author) / Buelt, Allison (Co-author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Division of Teacher Preparation (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
This study was conducted to observe the effects of vitamin C supplementation upon the expression of sICAM-1 in asthmatic subject. Two groups were created, each with a sample size of 4 subjects. One group was the vitamin C group (VC) and the other was the placebo group (PL). The study was analyzed through observing concentrations of biomolecules present within samples of blood plasma and nasal lavages. These included vitamin C, sICAM-1 expression, and histamine. The following P-values calculated from the data collected from this study. The plasma vitamin C screening was p=0.3, and after 18 days of supplementation, p=0.03. For Nasal ICAM p=0.5 at Day 0, p=0.4 at Day 4, and p=0.9 at Day 18. For the Histamine samples p=0.9 at Day 0 and p=0.9 at Day 18. The following P-values calculated from the data collected from both studies. The plasma vitamin C screening was p=0.8, and after 18 days of supplementation, p=0.03. The change of vitamin C at the end of this study and the combined data both had a P-value that was calculated to be lower than 0.05, which meant that this change was significant because it was due to the intervention and not chance. For Nasal ICAM samples p=0.7 at Day 0, p=0.7 at Day 4, and p=1 at Day 18. For the Histamine p=0.7 at Day 0 and p=0.9 at Day 18. This study carries various implications although the study data was unable to show much significance. This was the second study to test this, and as more research is done, and the sample size grows, one will be able to observe whether this really is the mechanism through which vitamin C plays a role in immunological functions.
ContributorsKapadia, Chirag Vinay (Author) / Johnston, Carol (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12