Browsing by Author "Gao, Xiaolian"
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Item Aptamers and Phage as Tools for the Development of Novel and Sensitive Point-of-Care Diagnostics(2014-08) Adhikari, Meena 1979-; Willson, Richard C.; Fox, George E.; Gao, Xiaolian; Williams, Cecilia M.; Bikram, MalavosklishImmunochromatographic lateral flow assays (LFAs) represent a well-established point-of-care diagnostic analytical method for the primary testing of diverse samples. The sensitivity of conventional LFAs that use the standard colored nanoparticles as reporters lags behind the more elaborate laboratory techniques, such as plaque counting, PCR or ELISA. To address this issue, we have introduced an innovative approach that utilizes functionalized M13 bacteriophage nanoparticles as reporters in the lateral flow diagnostic system. M13 phage offers a multitude of binding sites on its major coat protein pVIII for reporter enzymes, and for affinity agents specific for the target of interest. Furthermore we employed SAM-AviTag phage, derivatives of phage M13 in which the N-terminus of the tail protein pIII is replaced by the enzymatically-biotinylatable AviTag peptide (GLNDIFEAQKIEWHE). The lysine residue (K) in the AviTag is a substrate for biotinylation by the E. coli biotin ligase (birA) enzyme. Using streptavidin or Neutravidin, any biotinylated affinity agent can then easily be linked to these enzymatically-biotinylated phage particles. Viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). These particles were successfully integrated into a lateral-flow immunochromatographic assay detecting MS2 virus, a model for viral pathogens. The limit of detection of the assay was 10^4 MS2/mL, 1000-fold more sensitive than the conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments. Aptamers are short, library-selected nucleic acid molecules that can recognize and bind to pre-selected targets with high affinity and selectivity. They have been used as recognition elements in a variety of applications. We screened for DNA aptamers specific to the murine anti-lysozyme antibody, HyHEL-5. During the validation process, however, the originally selected aptamers did not show successful binding. As an alternative, literature DNA aptamers were employed to construct phage displaying aptamers and HRP, which were used as LFA reporters. We describe the first modification of bacteriophage particles with aptamers as bio-recognition elements, and demonstrate their use in ultrasensitive lateral flow assays detecting IgE and PBP2a (Penicillin binding protein) of Staphylococcus aureus.Item Binding and Signaling Differences between Prostaglandin E1 and E2 Mediated by Prostaglandin E Subtype Receptors(2010-08) Chillar, Annirudha; Ruan, Ke-He; Chow, Diana Shu-Lian; Gao, Xiaolian; Williams, Louis; Das, JoydipProstaglandin E1 (PGE1) and E2 (PGE2) are ligands for the prostaglandin E2 receptor (EP) family, which consists of four subtype receptors, designated as EP1, EP2, EP3 and EP4. Interestingly, PGE2 mediates inflammation whereas PGE1 acts as an anti-inflammatory factor. However, the molecular basis of their opposite actions on the same set of EP receptors is poorly understood. To study the ligand recognition differences, a potential high throughput mutagenesis and constrained peptide was used. A peptide constrained to a conformation of the second-extracellular loop of human prostaglandin-E2 (PGE2) receptor subtype 3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE2 were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [3H]PGE2. This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE2 recognition. The mutant S211L was able to give a calcium signal with PGE1, but not with PGE2. This implied that the corresponding residues in other subtype receptors could be important in distinguishing the different configurations of PGE2 and PGE1 ligand recognition sites. Direct transfection of point mutants in the EP1 receptor extracellular loop (using PCR products) was evaluated in HEK293 cells. Twenty-four EP1 extracellular loop mutants (alanine scan) were generated using phosporylated primers and ligase. The PCR product was directly transfected into HEK293 cells and the [3H]-PGE2 binding and PGE1 and PGE2 calcium signaling assay evaluated. Three mutants, A104G, P105A and P184A, showed reduced [3H]PGE2 binding, but could not differentiate between PGE1 and PGE2 in the calcium signaling assay. However, we propose that this novel high throughput mutagenesis approach using direct PCR product transfection can be integrated into a high throughput screening machine in the future. The PGE1 and PGE2 binding affinity on the four human recombinant EPs expressed in the live HEK293 as stable cell lines was determined by [3H]PGE2 binding. The PGE1 and PGE2 signaling on the four EPs was determined by the calcium (Ca2+) and cyclic AMP signaling. The Kd for [3H]PGE2 was calculated using saturation kinetic experiments. The IC50 of PGE2 and PGE1 were calculated from [3H]PGE2 displacement experiments using cold PGE2 and PGE1, respectively. PGE2 showed higher affinity or preference for EP3 and EP4 as compared to that of EP1 and EP2. PGE1 also showed a higher Ca2+ signal in EP1 as compared to that of PGE2. There was a two-log concentration difference between PGE2 and PGE1 for generation of Ca2+ signal in EP4. There was no difference in cAMP accumulation with PGE1 and PGE2. Leukotriene C4/D4/E2 levels were higher in the EP1 stable cell line upon stimulation with PGE2, but not PGE1. An anti-inflammatory molecule, 20 hydroxy lipoxin B4, peak was observed using mass spectroscopy with PGE1 and not PGE2. We also used a newly engineered hybrid enzyme (COX-2-10aa-mPGES-1) linking COX-2 and mPGES-1 together thus adopting the full biological activity of COX-2 and mPGES-1 in directly converting AA to PGE2. This enzyme was genetically introduced into HEK293 cells. These cells expressing the COX-2-10aa-mPGES-1 were producing higher level of PGE2 using endogenous AA as confirmed by LC/MS analysis. A new mouse model of cancer was developed by subcutaneous injection of these cells into Balb/c/nu/nu mice. A 100% (8 out 8) occurrence rate of cancer mass was detected in these cells. In contrast, 30% occurrence of cancer mass were determined for the groups of the cells co-expressing the individual COX-2 and mPGES-1. The presence of EP1 and EP2 stable cell line growth around these tumor masses confirmed their involvement in cancer. In conclusion, the Ca2+ signal indicated that the EP1 is likely the dominant and ligand-differentiating receptor in terms of signaling in tissues that co-express the EPs (cancer cells). PGE2 is likely to cause inflammation through leukotrienes and PGE1 is likely to be anti-inflammatory due to its ability to produce Lipoxin B4. High throughput mutagenesis for producing multiple-point mutations using direct PCR product transfection is a promising new method for the future. The experiments on nude mice indicated that the sole coupling of COX-2 to mPGES-1 is a powerful cancer-advancing factor, which implies that the coupling of COX-2 to mPGES-1 is a promising target for anti-cancer drug development. EP1 and EP2 receptors were identified as the likely receptors, to induce cancer. This study provides a molecular basis to understand the biological functions of PGE1 and PGE2 through their binding and signaling properties.Item Dual Impacts of Newly Engineered COX-PGIS Hybrid Enzyme on Cardiovascular and Neuronal Protection(2018-05) Ling, Qinglan; Ruan, Ke-He; Aronowski, Jaroslaw A.; Das, Joydip; Eikenburg, Douglas C.; Gao, XiaolianStatement of the problem: Non-steroidal anti-inflammation drugs (NSAIDs) are widely used for varieties of inflammatory conditions by inhibiting COX pathway and decreasing the production of downstream prostaglandins, such as PGE2 and PGI2. As neuroinflammation may lead to Alzheimer’s disease (AD), NSAIDs have also been suggested as a prevention or treatment for AD. However, clinical studies did not show effectiveness. To further understand the mechanisms and eventually achieve the goal, it is important to understand the crosstalk of prostanoids in the hippocampus, a major brain area impacted in AD. So far, a major obstacle in that was to mimic the biosynthesis of each prostanoid. Thus, this study intended to accomplish three aims. Specific aim 1: To study the crosstalk of prostanoids in a neuronal cell line by using hybrid enzymes to redirect the synthesis of prostanoids to PGE2 and PGI2, respectively. Specific aim 2: To investigate the crosstalk of prostanoids in primary hippocampal neurons. Specific aim 3: To examine the dual impacts of newly engineered COX-PGIS hybrid enzyme on cardiovascular and neuronal protection in vivo. Procedure or methods: Here, we created Single-Chain Hybrid Enzyme Complexes (SCHECs), COX-2-10aa-mPGES-1 and COX-1-10aa-PGIS, which could control cellular AA metabolites to the desired PGE2 or PGI2, respectively. By using the two hybrid enzymes, as well as synthetic PGE2 and PGI2 analogues, we investigated the crosstalk of PGE2 and PGI2 in hippocampal neuronal cell line, primary hippocampal neurons, and in vivo Alzheimer’s disease model. Results: The hippocampal neurons expressing SCHECs redirected AA metabolism to a PGI2, or PGE2. Overproduced PGI2 exerted survival protection and resistance to Aβ-induced neurotoxicity. The protection mediated by PGI2 might be through IP receptor. Our PGI2-producing transgenic mice exhibited resistance to AA-induced thrombotic stroke and angiotensin-II-induced vascular constriction. The hybrid of PGI2-producing transgenic mice and AD mice showed restored long-term memory in two behavioral tasks. Conclusions: These findings suggested that the vascular mediators, PGI2 and PGE2, exerted significant regulatory influences on neuronal protection (by PGI2), or damage (by PGE2) in the hippocampus, and raised a concern that the widely uses of aspirin in cardiovascular diseases may exert negative impacts on neurodegenerative protection.Item Effect of Selective Up regulation of Prostacyclin Synthesis On Its Signaling And Vascular Protection(2011-08) Mohite, Anita; Ruan, Ke-He; Bond, Richard A.; Williams, Louis; Gao, Xiaolian; Yu, RongProstacyclin (PGI2) is a very important endogenous vascular protector. It provides vascular protection through actions like vasodilatation, platelet aggregation and vascular smooth muscle cell proliferation inhibition and smooth muscle differentiation regulation. Therefore since decades, there have been many ongoing attempts to use PGI2 to treat vascular disorders such as pulmonary arterial hypertension, peripheral artery disease and thrombosis. However, the short half-life of PGI2 has limited its therapeutic potential. PGI2 is synthesized endogenously from arachidonic acid using enzymes cyclooxygenase isoform 1/2(COX-1/2) and prostacyclin synthase (PGIS). Single gene therapy with either COX or PGIS through adenoviral vectors does not specifically overproduce PGI2 and also is associated with inflammatory reactions. Thus to address these problems, our lab engineered a single hybrid gene (COX-1-10aa-PGIS) by fusing COX-1 with PGIS through transmembrane linker of 10 amino acids (10aa). Adipose tissue derived cells have been used therapeutically in several vascular disorders due to their regenerative properties. Thus we isolated and cultured mature adipocytes (MA) from mouse adipose tissue and called it as prostanoid synthesizing fat cells (PSFC). Further, we engineered a novel adipose tissue-derived cell that constantly produces PGI2, through transfecting of the engineered cDNA of the hybrid enzyme (human COX-1-10-aa-PGIS), which has superior triple catalytic functions in directly converting arachidonic acid into PGI2. The gene-transfected cells were further converted into a stable cell line, in which the cells constantly expressed the hybrid enzyme and were capable of overproducing specifically PGI2 and thus we called this cell line as PGI2 producing PSFC (PGI2-PSFC). When the results of the HPLC activity assay and LC/MS/MS were compared between just vector transfected (PSFC) and COX-1-10aa-PGIS gene transfected cells (PGI2-PSFC), it was observed that the majority of the endogenous AA metabolism shifted from that of unwanted PGE2 (in PSFC) to that of the preferred PGI2 (in PGI2-PSFC) with a PGI2/PGE2 ratio change from 0.03 to 25. The PGI2-producing cell line not only exhibited an approximate 50-fold increase in PGI2 biosynthesis, but also demonstrated superior anti-platelet aggregation in vitro, and increased reperfusion in the mouse ischemic hindlimb and thrombogenesis models in vivo. PGI2 regulates functions like vascular smooth muscle cell differentiation and proliferation and apoptosis. These actions are mediated through the PGI2 receptor (IP) and nuclear receptor, peroxisome proliferator-activated receptors (PPAR). MicroRNAs (miRNAs), which are negative regulators of gene expression, also regulate similar functions like PGI2. PGI2 regulates expression of genes by signaling through PPARs. Thus to find out if PGI2 regulates gene expression by regulating miRNA expression by signaling through the IP and PPAR, a miRNA microarray analysis of the PGI2-PSFCs and PSFCs was performed. Analysis of the results obtained from miRNA study, revealed that PGI2 is involved in regulating cellular microRNA (miRNA) expression through its receptors in the mouse adipose tissue-derived primary culture cell line overexpressing COX-1-10aa-PGIS (PGI2-PSFC). The miRNA microarray analysis of the PGI2-PSFC revealed a significant up-regulation (711, 148b, and 744) and down-regulation of miRNAs of interest, which were reversed by antagonists to the IP and PPARγ receptors. Furthermore, we also found that the insulin-mediated lipid deposition was inhibited in the PGI2-PSFCs. The study also initiated a discussion, which suggested that the endogenous PGI2 inhibition of lipid deposition in adipocytes could involve miRNA-mediated inhibition of expression of the targeted genes specifically of Akt1. To translate the results of in vitro study, a transgenic mouse permanently expressing the COX-1-10aa-PGIS gene was generated. This genetic mouse animal model was used to study the effect of COX-1-10aa-PGIS overexpression on PGI2 production in vivo and as well as its vascular protective effect. The transgenic mice was genotyped using polymerase chain reaction and further bred to develop a colony of homozygous transgenic mice. Quantitative PCR was performed to distinguish between the hemizygous and homozygous mice. PCR and imunoblot analysis of all the organs and tissues of mice revealed the expression of COX-1-10aa-PGIS in adipose tissue, brain, heart and uterus. LC/MS analysis of urine and plasma revealed approximately five-folds increase in PGI2 biosynthesis permanently in vivo. The animals showed strong resistance to photochemical induced thrombosis. They produced healthy pups with a an increase in live births when compared to the wild type without significant adverse effects in development and functions of major organs including brain, heart, kidneys. Tail cuff method was used to measure blood pressure of the mice which was similar to that in the wild type. Thus PGI2-PSFCs (therapeutic cells) can be used as cell therapy as an alternative to adenoviral gene transfer to treat ischemic and vascular conditions. The cells, which have an ability to increase the biosynthesis of the vascular protector, PGI2, while reducing that of the vascular inflammatory mediator, PGE2, provide a dual effect on vascular protection, which is not available through any existing drug treatments. Thus, the current finding has potential to be an experimental intervention for PGI2-deficient diseases, such as pulmonary arterial hypertension. PGI2 mediated gene expression regulation through miRNA expression regulation has unraveled a novel signaling mechanism for PGI2. This signaling could exist in broad pathophysiological processes involving PGI2 (i.e. apoptosis, vascular inflammation, cancer, embryo implantation, and obesity). The results of the transgenic mice overexpressing COX-1-10aa-PGIS and producing PGI2 provides the first evidences that risk of stroke and heart diseases could be reduced by the human transgene, as well as the insight into the possible future transgenic medicine to prevent and reduce the genetic risk of heart disease.Item Exploring estrogen receptor gene regulatory mechanism in breast cancer(2013-08) Katchy, Anne Chinenye 1984-; Williams, Cecilia M.; Schwartz, Robert J.; Webb, Paul; Gao, XiaolianEstrogen has vital roles in development and maintenance of mammary gland and supports the growth of the majority of primary breast cancer. It carries out its function through the estrogen receptors (ER): ERα and ERβ. In this dissertation, we focused on identifying the functional and genome wide effects of both receptors in breast cancer cell lines (T47D and MCF7). First, we aimed at identifying microRNAs (miRNAs) that are significantly associated with normal or disrupted estrogen signaling in breast cancer cells, and are regulated by ERα and ERβ. Despite the fact that 24h estrogen treatment results in strong changes to expression of about 900 protein-coding transcripts, we found no significant changes in mature miRNA expression levels by 17β-estradiol (E2)-activated ERα in neither T47D nor MCF7 breast cancer cells. On the other hand, when studying the effects of exogenously expressed ERβ, we identified miR-135a, miR-21, miR-200c, and miR-522, among others, as being regulated. Most of the ERβ effects were ligand-independent, but we observed a significant response to E2 in the MCF7-ERβ cells. Also, the MCF7-ERβ showed enhanced stem cell abilities in comparison to the MCF7-Control cells as illustrated by increased mammospheres formation during several generations. Secondly, we aimed at investigating environmental factors and its effect on the risk for breast cancer. We identified the gene expression response for each of these compounds: BPA, genistein (Gen), E2, and soy formula extract (SF) in MCF7 cells, and found that each regulated similar genes in the same manner. Many target genes regulated by BPA, Gen, and SF, were involved in important biological processes identical to those of estrogen. Investigation of non-ERα mediated regulations for these EDCs suggested that these compounds may regulate gene transcription solely through ERα, in the cells and doses used. Furthermore, we found that these compounds acted together in a sub-additive manner, and tumor clustering with the gene expression profile of these EDC compounds revealed a significantly lower disease free survival. Altogether, the data presented in this dissertation would aid in increasing the knowledge of early risk factors for breast cancer. Our work provides data for future use of the estrogen receptors in clinical applications by providing new candidates for pharmaceutical drug development, as well as, biomarkers for diagnosis and prognosis.Item Impact of Dicer on the Embryonic Stem Cell Epigenome and Androgen Mediated AMPK-PGC-1α Signaling in Prostate Cancer(2013-08) Tennakoon, Jayantha 1969-; Gunaratne, Preethi H.; Frigo, Daniel E.; Wells, Dan E.; Gao, Xiaolian; Zwaka, Thomas P.What mechanisms govern a cellular phenotype is a fascinating question for which answers are yet being sought. The work presented in this dissertation is an effort to address two fundamental questions, which relate to cellular transition of pluripotent stem cells to a differentiated state and the ability of prostate cancer to have increased proliferative potential. Dicer is an evolutionary conserved RNAse III type endoribonuclease enzyme, which plays a pivotal role in the biogenesis of microRNAs and silencing RNAs (siRNAs). Within the first chapter herein using in vitro cultures of embryonic stem cells, I show that loss of Dicer leads to changes in the ES cell epigenome resulting in a shift in transcriptionally favorable versus transcriptionally unfavorable histone modifications and thereby affect gene expression critical for precise cellular differentiation. In the second chapter, employing a combination of molecular biological and modern metabolomics approaches I show that androgen signaling deregulated in almost all forms of metastatic prostate cancers can lead to increased mitochondrial biogenesis and ATP production affording a distinct proliferative advantage. The underlying mechanism is linked to androgen mediated AMPK- PGC-1α signaling which results increased oxidative capacity in addition to elevated glycolytic capacity quite well established in numerous types of cancers. The pathway uncovered provides an interesting option for targeted therapeutics of prostatic cancers that are particularly resistant to androgen ablation therapies. Finally in the third chapter I show the significance of Dicer in maintaining expression levels of developmentally critical mammalian imprinted genes. The combined results of this thesis provide mechanistic insights into developmentally critical cellular pathways in embryonic stem cells and cancer having high potential to be manipulated in stem cell and molecular intervention based therapeutics.Item MICRORNA: PROFILING AND FUNCTIONAL IMPLICATIONS IN CANCER AND METABOLISM(2012-12) Wang, Xin 1984-; Gao, Xiaolian; Sater, Amy K.; Williams, Cecilia M.; Liu, Chang-GongMicroRNAs (miRNAs) are a class of ~22 nt short, non-coding RNAs that post-transcriptionally regulate target mRNA expression. To date, ~2,000 mature miRNAs have been identified in humans and they are estimated to regulate about 50% of human genes. miRNAs, due to their ubiquitous target distribution, contribute to diverse processes including cell development, proliferation, differentiation, apoptosis, and metabolism. Dysregulation of miRNA expression has been reported in various cancers and metabolic disorders. miRNA are also implicated in the initiation and progression of those diseases. In my dissertation, I studied the differentially expressed (DE) miRNAs upon prostaglandin E2 (PGE2) stimulation in prostate cancer cells (PC-3). Concurrently I examined mRNA expression profile of the PC-3 system and determined anticorrelated miRNA:mRNA pairs. The DE miRNAs and their putative targets were affected by the induction of PGE2. They were suggested to be involved in PGE2 dysregulated signaling pathways in PC-3 prostate cancer. In the second part of the thesis work, I identified a set of adipose-enriched miRNAs from porcine tissues samples and verified that these miRNAs were conserved in humans. Adipose-enriched miRNAs were reported to be involved in metabolism, inflammation responses, and tumorigenesis. The analysis results of my thesis experiments suggested adipose-enriched miRNAs may have a potential role in connecting obesity, inflammation, and cancer. It is hoped that the understanding of the molecular basis in cancer and metabolic disorders on the miRNA level will provide new diagnostic targets and therapeutic targets for the diseases.Item Nucleotide modification at the ?-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase(Nucleic Acids Research, 1/1/2005) Mulder, Brent A.; Anaya, Steve; Yu, Peilin; Lee, Keun Woo; Nguyen, Anvy; Murphy, Jason C.; Willson, Richard C.; Briggs, James M.; Gao, Xiaolian; Hardin, Susan H.The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their ?-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the ?-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.Item Omics-Scale Bioinformatics Technology and Methods: from Data to Information(2014-12) Chen, Haosi 1987-; Gao, Xiaolian; Briggs, James M.; Williams, Cecilia M.; Yu, FuliOmics-scale bioinformatics is an emerging discipline of science that plays an essential role in analyzing and interpreting large scale biological data. In this thesis, I developed three different omics-scale bioinformatics methods to facilitate the studies of human miRNAs, synthetic DNA oligo library and histone post-translational modifications (PTMs), respectively. MiRNAs, which are involved in various biological processes by regulating multiple genes, have been an area of research drawing intensive interest in the recent two decades. There exists an enormous amount of miRNA related information, and how to effectively mine the valuable information embedded in the large volume of literature has become an urgent problem. Because each of the existing online databases includes only partial information about human miRNAs, I created a comprehensive web-based resource ‘miRFocus’ for conveniently retrieving extensive and comprehensive human miRNA information and conducting pathway and Gene Ontology (GO) term enrichment analysis. Current next-generation sequencing (NGS) technologies mainly focus on genome or transcriptome sequencing analysis and none of the existing NGS methods is suitable for high resolution nucleobase-specific analysis of libraries of synthetic oligonucleotides, which are used as materials for engineering long DNA fragments in synthetic biology applications. To meet such requirements, I developed an algorithm and software tool for analyzing synthetic oligo libraries. This approach is composed of two-step quality control and Bowtie2-based sequence alignments. It is proved that such a method successfully assessed the efficiency of etMICC-based error-removal method on synthetic oligos of different lengths and identified that etMICC columns has higher binding affinity with gap error structure than substitution error structure. Epiproteomics examines diverse PTMs, such as histone methylation. However, traditional methods of studying histone PTMs are expensive in cost, labor and time. I developed a histone peptide array (hPepArray) for analyzing activities of cellular histone methyltransferases (HMTs). Lysine-containing peptides of hPepArray are directly generated from 10 histone proteins. In the hPepArray, two known methylation sites H3K122 and H4K59 are verified and one possible methylation site H2A-K74 is identified. The experimental results demonstrate that hPepArray and the method of analysis offer a high-throughput epiproteomic tool to assay activities of HMTs in nuclear lysates.Item Proteomic Analysis of Post-Translational Modifications and Signaling Pathways in Pancreatic Cancer(2019-12) Vo, Hiep T. 1990-; Gao, Xiaolian; Bawa-Khalfe, Tasneem; Frigo, Daniel E.; Gunaratne, Preethi H.Signaling pathways mediated by receptor tyrosine kinases (RTKs) and protein kinases play essential roles in cellular physiology. Src Homology 2 (SH2) domains bind to specific phosphotyrosine motifs and link activated RTKs to several downstream pathways. Studying SH2 domain-containing proteins implicated in cancer signaling networks under given therapies can yield significant insight into the molecular basis of drug responses, thus having valuable clinical applications. Here, we demonstrate a microfluidic, peptide-microarray biochip technology (μPepArray™) for the label-free and multiplex detection of the endogenous SH2 domain proteins and signaling pathways from total protein lysate. The biochip contains 3968 reaction chambers, where the immobilized phosphopeptides serve as the molecular probes to capture present SH2 domain proteins. Using pancreatic cancer cells treated with epidermal growth factor receptor inhibitors (EGFRis), we identified the differentially expressed SH2 domain proteins such as SHP2, PLCG1, PI3K, and therefore the downstream phosphotyrosine-mediated signaling events occurred in response to treatments. Our findings revealed a signaling compensation between the EGFR/PI3K/Akt and mTORC2/PKCα/ERK pathways as a mechanism of EGFRi responses and potential resistance in BxPC-3 cells. We propose that the proteomic information provided by μPepArray™ technology could hold clinical significance by assisting in the cancer biomarker identification and guiding future treatment decisions. In the second approach of this dissertation, we utilize μPepArray™ technology for the high-throughput identification of histone SUMOylation. SUMOylation is a dynamic post-translational modification (PTM) process which covalently attaches a small ubiquitin-like modifier (SUMO) to a protein substrate. This modification alters protein functions and activities, ultimately affecting cellular responses and homeostasis. Histones play essential roles in epigenetic regulations. Different histone PTMs directly dictate chromatin structure and transcriptional activities. Identifying histone SUMOylation and specific lysine residues that are SUMOylated has been challenging due to limitations in technology. Using a customized library of lysine peptides derived from histone sequences, we have identified multiple histone SUMO modification sites as well as peptides that bind non-covalently to SUMO on the microarray chip. We propose that μPepArray™ technology offer a rapid screening assay to determine protein post-translational modifications, which will provide significant insights into PTM substrate specificity and their functions.Item Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts(Nature Methods, 3/2/2014) Sripakdeevong, Parin; Cevec, Mirko; Chang, Andrew T.; Erat, Michele C.; Ziegeler, Melanie; Zhao, Qin; Fox, George E.; Gao, Xiaolian; Kennedy, Scott D.; Kierzek, Ryszard; Nikonowicz, Edward P.; Schwalbe, Harald; Sigel, Roland K. O.; Turner, Douglas H.; Das, RhijuStructured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating 1H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6–2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.Item The Endogenous Metabolite 5-Methoxytryptophan as a Potential Novel Therapy for Pancreatic Cancer(2017-08) Murdoch, Emma E.; Ruan, Ke-He; Dixon, Richard A. F.; Williams, Louis; Das, Joydip; Gao, XiaolianPancreatic adenocarcinoma is a highly aggressive cancer, and due to a lack of early detection methods, is often diagnosed in the late stages of the disease. One reason for the poor prognosis in pancreatic cancer is the repopulation of tumors, which is driven by pathological cyclooxygenase-2 production. The L-tryptophan metabolite 5-MTP has previously been shown to reduce COX-2 transcription via the p300 histone acetyltransferase. In a scratch test assay using the PANC-28 cell line, we demonstrated that PANC-28 cell invasion could be reduced by treatment with 5-MTP at the micromolar level. This project included molecular modeling of 5-MTP at the PGE2 receptor EP3. 5-MTP showed a strong binding score, in a similar location to that of the endogenous prostaglandins. A cell counting kit was used to measure cell survival following 5-MTP administration, and showed that 5-MTP was non-toxic to cells. Given our results, it is possible that 5-MTP may have a beneficial role in pancreatic cancer. Future studies will include biochemical and ligand binding assays to build upon these findings.Item To examine the combinatorial therapeutic effects of EGFR inhibitor and mTORC2 inhibitor for treatment of pancreatic cancer(2019) Modi, Paulomi; Vo, Henry; Robertson, CeliseThe role of protein expression and signaling pathways in cancer cells going under drug treatment has become apparent. It could yield significant insight into developing therapeutic response/ treatment of the diseases. A contributor of pancreatic cancer cell advancement and growth is the inflection of the protein expression and cell signaling pathway. This is a result of the mis-synchronization of protein kinase activity. Transformation of oncogenes can be caused due to mutations in kinase encoded genes. These genes are frequently found in cancer. Kinase inactivation occurs by the actions of these molecules as they are competing with ATP binding to the enzyme allosteric sites. Hence, they disturb cellular signaling pathways resulting in cancer cell growth and survival to deter. As a result, for this experiment, the is target mTORC2 pathway with inhibitors along with various EGFR inhibitors. MTT cell proliferation data represented with the Q curve shows a significant decrease in cell viability when treated with the combination of EGFR and mTORC1/mTORC2 inhibitors. The EGFR inhibitors cause a reduced downstream regulation of PI3K/Akt/mTOR signaling by inhibiting the EGFR phosphorylation. Afatinib is shows inhibitions of EGFR and HER2. Erlotinib and AZD9291 only inhibit EGFR.AZD8055 (mTORC1/mTORC2 inhibitor) that prevents the growth pancreatic cancer cells in combination with EGFR inhibitors (Erlotinib, Afatinib, AZD9291).Combinatorial treatment with EGFRi (Erlotinib, Afatinib, or AZD9291) and mTORi (AZD8055) improved anti-proliferative effects on BxPC-3 cell as compared to EGFRi treatment alone. This combination is shown to be synergetic by Chou and Talalay method, showing high synergetic drug combination at and above IC50. Furthermore, the relative importance of this study, it is important to look at the proteomic complexity and the respective modulating signaling networks. They are derived from alterations in the oncogenes. The therapeutic treatments and resistant mechanisms needed for these alterations have not been fully understood. This presents us with the critical requirement- to minimize the adverse effects of the therapeutic responses and increase drug resistance towards the cancer cells. Thus, the ultimate goal is to enhance treatment efficacy.Item Understanding Structure and Dynamics of PTEN and its Possible Genotype-Phenotype Correlations in Endometriosis and Cancer(2016-12) Smith, Iris Nira 1978-; Briggs, James M.; Zhang, Weihua; Heard, Michael J.; Gao, XiaolianThe phosphatase and tensin homolog deleted on chromosome 10, (PTEN) gene encodes a tumor suppressor phosphatase frequently mutated in various human cancers. Somatic missense mutations of PTEN have recently been found in patients with endometriosis, endometrial cancer, and ovarian cancer. Here we present the first computational analysis of 13 somatic missense PTEN mutations to assess a possible genotype-phenotype correlation in endometriosis and cancer. We posit PTEN’s active site defines a possible mutation-driven allosteric region wherein a subset of mutations correlate with endometriosis, endometrial cancer, and ovarian cancer. Our data suggest that mutations within the active site disrupt the structural stability, electrostatic interaction, global dynamics and the structural communication pathway, likely contributing to the aforementioned phenotypes. Multiple in silico prediction methods were utilized to calculate protein structural stability changes produced by each mutation; decreases in protein structure stability were seen in each mutation with an increase in dynamics across the phosphatase-C2 domain interface of R130G/L/Q and R173C/H mutations. To assess the impact on intrinsic and global dynamics, elastic network models (ENMs) were employed demonstrating changes from wild-type “hinge-bending” to “zipper-like” global motions induced by each mutation. All-atom molecular dynamics (MD) simulations revealed large conformational changes that affect the global dynamics of the active site loops and the CBR3 loop in the C2 domain. Interestingly, mutations G36E/R, C124S, G129R, R130L/Q, R173C/H, and V191A dramatically affected the principal motions of the active site loops and inter-domain interface. Overall, the global dynamics induced by each mutation effects reveal unique long-range perturbations that may impair PTEN’s function. We further investigated structural communication within each mutant system using protein structure network (PSN) analysis and found that R130 and R173 play critical roles in controlling salient communication pathways suggesting a compelling interplay between the two positions involving a potential mutation-driven allosteric interface. The results of this research provide a greater understanding of the mechanistic role of mutated PTEN associated with endometriosis and cancer. It is our hope that these results will aid in a better clinical-molecular classification of the resulting phenotypes allowing for translation into improved diagnostic and therapeutic approaches.