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- All Subjects: Biochemistry
- Genre: Academic theses
- Creators: LaBaer, Joshua
- Member of: Theses and Dissertations
- Status: Published
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
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
Exposure of liquid biospecimens like plasma and serum (P/S) to improper handling and storage can impact the integrity of biomolecules, potentially leading to apparent quantitative changes of important clinical proteins. An accurate and quick estimate of the quality of biospecimens employed in biomarker discovery and validation studies is essential to facilitating accurate conclusions. ΔS-Cys-Albumin is a marker of blood P/S exposure to thawed conditions that can quantitatively track the exposure of P/S to temperatures greater than their freezing point of -30 C. Reported here are studies carried out to evaluate the potential of ΔS-Cys-Albumin to track the stability of clinically important analytes present in P/S upon their exposure to thawed conditions. P/S samples obtained from both cancer-free donors and cancer patients were exposed to 23 C (room temperature), 4 C and -20 C degrees, and the degree to which the apparent concentrations of clinically relevant biomolecules present in P/S were impacted during the time it took ΔS-Cys-Albumin to reach zero was measured. Analyte concentrations measured by molecular interaction-based assays were significantly impacted when samples were exposed to the point where average ΔS-Cys-Albumin fell below 12% at each temperature. Furthermore, the percentage of proteins that became unstable with time under thawed conditions exhibited a strong inverse linear relationship to ΔS-Cys-Albumin, indicating that ΔS-Cys-Albumin can serve as an effective surrogate marker to track the stability of other clinically relevant proteins in plasma as well as to estimate the fraction of proteins that have been destabilized by exposure to thawed conditions, regardless of what the exposure temperature(s) may have been. These results indicated that P/S exposure to thawed conditions disrupts epitopes required for clinical protein quantification via molecular interaction-based assays. In continuation of this theme, a spurious binding event between two clinically important proteins, Apolipoprotein E (ApoE) and Interferon- (IFN) present in human plasma under in vitro experimental conditions is also reported. The interaction was confirmed to be evident only when ApoE was expressed in vitro with a Glutathione-S-Transferase (GST) fusion tag. Future steps required to find the exact manner in which the GST fusion tag facilitated the association between ApoE and IFNγ are discussed with emphasis on the possible pitfalls associated with using fusion proteins for studying novel protein-protein interactions.
ContributorsKapuruge, Erandi Prasadini (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2021
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
ContributorsHanavan, Paul D (Author) / Lake, Douglas (Thesis advisor) / LaBaer, Joshua (Committee member) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Phenotypic and molecular profiling demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and the mutation frequency in basal-like subtype is as high as 80% and co-exists with several other somatic mutations in different genes. It was hypothesized that tumor heterogeneity is a result of a combination of neo-morphic functions of specific TP53 driver mutations and distinct co-mutations or the co-drivers for each type of TP53 mutation. The 10 most common p53 missense mutant proteins found in breast cancer patients were ectopically expressed in normal-like mammary epithelial cells and phenotypes associated with various hallmarks of cancer examined. Supporting the hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and polarity was observed in the mutants compared to wildtype p53 expressing cells. The missense mutants R248W, R273C and Y220C were most aggressive. Integrated analysis of ChIP and RNA seq showed distinct promoter binding profiles of the p53 mutant proteins different than wildtype p53, implying altered transcriptional activity of mutant p53 proteins and the phenotypic heterogeneity of tumors. Enrichment and model-based pathway analyses revealed dysregulated adherens junction and focal adhesion pathways associated with the aggressive p53 mutants. As several somatic mutations co-appear with mutant TP53, we performed a functional assay to fish out the relevant collaborating driver mutations, the co-drivers. When PTEN was deleted by CRISPR-Cas9 in non-invasive p53-Y234C mutant cell, an increase in cell invasion was observed justifying the concept of co-drivers. A genome wide CRISPR library-based screen on p53-Y234C and R273C cells identified separate candidate co-driver mutations that promoted cell invasion. The top candidates included several mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, the combined approach of molecular profiling and functional genomics screen highlighted distinct sets of co-driver mutations that can lead to heterogeneous phenotypes and promote aggressiveness in cells with different TP53 mutation background, which can guide development of novel targeted therapies.
ContributorsPal, Anasuya (Author) / LaBaer, Joshua (Thesis advisor) / Roberson, Robert (Committee member) / Van Horn, Wade (Committee member) / Maley, Carlo (Committee member) / Arizona State University (Publisher)
Created2019
Description
Elucidation of Antigen-Antibody (Ag-Ab) interactions is critical to the understanding of humoral immune responses to pathogenic infection. B cells are crucial components of the immune system that generate highly specific antibodies, such as IgG, towards epitopes on antigens. Serum IgG molecules carry specific molecular recognition information concerning the antigens that initiated their production. If one could read it, this information can be used to predict B cell epitopes on target antigens in order to design effective epitope driven vaccines, therapies and serological assays. Immunosignature technology captures the specific information content of serum IgG from infected and uninfected individuals on high density microarrays containing ~105 nearly random peptide sequences. Although the sequences of the peptides are chosen to evenly cover amino acid sequence space, the pattern of serum IgG binding to the array contains a consistent signature associated with each specific disease (e.g., Valley fever, influenza) among many individuals. Here, the disease specific but agnostic behavior of the technology has been explored by profiling molecular recognition information for five pathogens causing life threatening infectious diseases (e.g. DENV, WNV, HCV, HBV, and T.cruzi). This was done by models developed using a machine learning algorithm to model the sequence dependence of the humoral immune responses as measured by the peptide arrays. It was shown that the disease specific binding information could be accurately related to the peptide sequences used on the array by the machine learning (ML) models. Importantly, it was demonstrated that the ML models could identify or predict known linear epitopes on antigens of the four viruses. Moreover, the models identified potential novel linear epitopes on antigens of the four viruses (each has 4-10 proteins in the proteome) and of T.cruzi (a eukaryotic parasite which has over 12,000 proteins in its proteome). Finally, the predicted epitopes were tested in serum IgG binding assays such as ELISAs. Unfortunately, the assay results were inconsistent due to problems with peptide/surface interactions. In a separate study for the development of antibody recruiting molecules (ARMs) to combat microbial infections, 10 peptides from the high density peptide arrays were tested in IgG binding assays using sera of healthy individuals to find a set of antibody binding termini (ABT, a ligand that binds to a variable region of the IgG). It was concluded that one peptide (peptide 7) may be used as a potential ABT. Overall, these findings demonstrate the applications of the immunosignature technology ranging from developing tools to predict linear epitopes on pathogens of small to large proteomes to the identification of an ABT for ARMs.
ContributorsCHOWDHURY, ROBAYET (Author) / Woodbury, Neal (Thesis advisor) / LaBaer, Joshua (Committee member) / Sulc, Petr (Committee member) / Arizona State University (Publisher)
Created2020
Description
Proteins are a large collection of biomolecules that orchestrate the vital
cellular processes of life. The last decade has witnessed dramatic advances in the
field of proteomics, which broadly include characterizing the composition, structure,
functions, interactions, and modifications of numerous proteins in biological systems,
and elucidating how the miscellaneous components collectively contribute to the
phenotypes associated with various disorders. Such large-scale proteomics studies
have steadily gained momentum with the evolution of diverse high-throughput
technologies. This work illustrates the development of novel high-throughput
proteomics platforms and their applications in translational and structural biology. In
Chapter 1, nucleic acid programmable protein arrays displaying the human
proteomes were applied to immunoprofiling of paired serum and cerebrospinal fluid
samples from patients with Alzheimer’s disease. This high-throughput
immunoproteomic approach allows us to investigate the global antibody responses
associated with Alzheimer’s disease and potentially identify the diagnostic
autoantibody biomarkers. In Chapter 2, a versatile proteomic pipeline based on the
baculovirus-insect cell expression system was established to enable high-throughput
gene cloning, protein production, in vivo crystallization and sample preparation for Xray diffraction. In conjunction with the advanced crystallography methods, this endto-end pipeline promises to substantially facilitate the protein structural
determination. In Chapter 3, modified nucleic acid programmable protein arrays
were developed and used for probing protein-protein interactions at the proteome
level. From the perspective of biomarker discovery, structural proteomics, and
protein interaction networks, this work demonstrated the power of high-throughput
proteomics technologies in myriad applications for proteome-scale structural,
functional, and biomedical research.
cellular processes of life. The last decade has witnessed dramatic advances in the
field of proteomics, which broadly include characterizing the composition, structure,
functions, interactions, and modifications of numerous proteins in biological systems,
and elucidating how the miscellaneous components collectively contribute to the
phenotypes associated with various disorders. Such large-scale proteomics studies
have steadily gained momentum with the evolution of diverse high-throughput
technologies. This work illustrates the development of novel high-throughput
proteomics platforms and their applications in translational and structural biology. In
Chapter 1, nucleic acid programmable protein arrays displaying the human
proteomes were applied to immunoprofiling of paired serum and cerebrospinal fluid
samples from patients with Alzheimer’s disease. This high-throughput
immunoproteomic approach allows us to investigate the global antibody responses
associated with Alzheimer’s disease and potentially identify the diagnostic
autoantibody biomarkers. In Chapter 2, a versatile proteomic pipeline based on the
baculovirus-insect cell expression system was established to enable high-throughput
gene cloning, protein production, in vivo crystallization and sample preparation for Xray diffraction. In conjunction with the advanced crystallography methods, this endto-end pipeline promises to substantially facilitate the protein structural
determination. In Chapter 3, modified nucleic acid programmable protein arrays
were developed and used for probing protein-protein interactions at the proteome
level. From the perspective of biomarker discovery, structural proteomics, and
protein interaction networks, this work demonstrated the power of high-throughput
proteomics technologies in myriad applications for proteome-scale structural,
functional, and biomedical research.
ContributorsTang, Yanyang (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen S (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2020