Matching Items (35)
Description
Pleiotrophin (PTN) is a cell-signaling protein in the human body that plays a pivotal role in the development of the central nervous system. It is known to have a high affinity for glycosaminoglycan (GAG), a type of linear polysaccharide. PTN has the ability to bind to a wide range of receptors, including receptor-type protein tyrosine phosphatase ζ (PTPRZ), a protein expressed in embryonic stem cells that regulates signals associated with survival, cell proliferation, and stem cell pluripotency. Several of these receptors are proteoglycans that carry GAGs, and the interaction between PTN and GAG has proven to be crucial to PTN’s functionality. Though PTN performs several important biochemical duties in normal cellular processes, this protein is upregulated in various cancer cell lines, primarily glioblastoma, an aggressive form of cancer that arises in the brain or spinal cord. The high levels of PTN expression in these forms of cancer may correlate to the cancer cells’ metastatic ability in the body. Determining how these PTN-GAG interactions form in cells is imperative for understanding how they may correlate to the development of cancer cell lines such as glioblastoma. However, due to the NMR signal degeneracy among the lysines in PTN, it is currently not possible to distinguish between lysines that have strong interactions with GAG and those that do not. To overcome this, pyrrolysyl-tRNA synthetase-mediated amber codon suppression is used to incorporate a single 15N-labeled lysine, Boc-lysine (Boc-K), at a specific position. This thesis seeks to optimize the systems and conditions needed to achieve amber codon suppression. The Origami B (DE3) strain is commonly used to achieve this, and demonstrates positive expression of PTN. The first aim of this project is to determine whether SHuffle® demonstrates enhanced expression of PTN and, therefore, incorporation of Boc-K. However, upon comparing PTN expression results, it was found that SHuffle® and Origami B(DE3) demonstrated similar levels of PTN expression. This project's second phase is focused on using C321.ΔA (Church) strain to evaluate differences in PTN expression compared to SHuffle® and Origami B(DE3). Expression testing indicated, however, that the expression of PTN in Church strain was inconclusive.
ContributorsKuchibhotla, Aditya (Author) / Wang, Xu (Thesis director) / Mills, Jeremy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2023-05
Description
Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
ContributorsXiao, Lu, Ph.D (Author) / Guo, Jia (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019
Description
Transient receptor potential vanilloid member 1 (TRPV1) is a membrane protein ion channel that functions as a heat and capsaicin receptor. In addition to activation by hot temperature and vanilloid compounds such as capsaicin, TRPV1 is modulated by various stimuli including acidic pH, endogenous lipids, diverse biological and synthetic chemical ligands, and modulatory proteins. Due to its sensitivity to noxious stimuli such as high temperature and pungent chemicals, there has been significant evidence that TRPV1 participates in a variety of human physiological and pathophysiological pathways, raising the potential of TRPV1 as an attractive therapeutic target. However, the polymodal nature of TRPV1 function has complicated clinical application because the TRPV1 activation mechanisms from different modes have generally been enigmatic. Consequently, tremendous efforts have put into dissecting the mechanisms of different activation modes, but numerous questions remain to be answered.
The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.
The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.
ContributorsKim, Minjoo (Author) / Van Horn, Wade D (Thesis advisor) / Wang, Xu (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
Description
There is increasing interest and demand in biology studies for identifying and characterizing rare cells or bioparticle subtypes. These subpopulations demonstrate special function, as examples, in multipotent proliferation, immune system response, and cancer diagnosis. Current techniques for separation and identification of these targets lack the accuracy and sensitivity needed to interrogate the complex and diverse bioparticle mixtures. High resolution separations of unlabeled and unaltered cells is an emerging capability. In particular, electric field-driven punctuated microgradient separations have shown high resolution separations of bioparticles. These separations are based on biophysical properties of the un-altered bioparticles. Here, the properties of the bioparticles were identified by ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities.
As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.
As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.
ContributorsLiu, Yameng (Author) / Hayes, Mark A. (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020
Description
Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins.
ContributorsBaravati, Bobby (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Van Horn, Wade (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2020
Description
The small mitogenic cytokine Pleiotrophin (PTN) is well-known for its roles in
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
ContributorsRyan, Eathen (Author) / Wang, Xu (Thesis advisor) / Yarger, Jeffery (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future.
ContributorsShen, Di (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2020
Description
Glycans are complex biological sugar polymers that are commonly found covalently attached to proteins, lipids, and lipoproteins. About 50% of all mammalian proteins are glycosylated. Aberrant glycosylation is a hallmark of most types of cancer, and glycosylation changes that occur in this disease are known to facilitate tumor development. In this dissertation, a bottom-up approach to glycomics, “glycan node analysis”, which is a method based on glycan linkage analysis that quantifies unique glycan features, such as “core fucosylation”, “α2-6 sialylation”, “β1-6 branching”, and “bisecting GlcNAc”, as single analytical signals by gas chromatography-mass spectrometry (GC-MS), was applied to cancer cell lines, antibodies, extracellular vesicles, and low density lipoproteins to understand the mechanisms leading to aberrant glycosylation in cancer, and to understand the role of blood plasma glycan sialylation in cancer immunity. Specific tumor antigens such as β1-6-branching, β1-4-branching, bisecting GlcNAc, antennary fucosylation, and Tn antigen (GalNAc-Ser/Thr), were found to be regulated by IL-6 in HepG2 cells; fewer glycan features were regulated by IL-1β. Additionally, neuraminidase enzyme treatment of alpha-1 antitrypsin IgG demonstrates how glycan node analysis can be used to detect relative changes in “α2-6-sialylation” along with corresponding increases in terminal galactose.
Extracellular vesicles (EVs) derived from metastatic and non-metastatic cancer cell lines displayed upregulated or downregulated expression of several specific glycan nodes, particularly 3-GlcNAc, which represents hyaluronic acid. EVs displayed several glycan features that distinguished them from the whole blood plasma glycome. These results were promising for developing new diagnostic strategies in cancer.
A “liquid phase permethylation” procedure for glycan node analysis that does not require spin columns was applied for the first time to whole biological specimens, and it demonstrated potential clinical utility in detecting specific tumor antigens. Significantly different glycan node profiles were found among three cancer cell lines and in peripheral blood mononuclear cells from healthy donors.
Changes in glycosylation and mechanisms regulating glycan changes were studied extensively in cancer cells. Subsequently, it is reported how glycosylation changes can have an impact in cancer immunity. A novel role for oxidized-desialylated low density lipoprotein in cancer immunity is reported, and its implications in cancer and atherosclerosis are discussed.
ContributorsAguilar Diaz de leon, Jesús Salvador (Author) / Borges, Chad R (Thesis advisor) / Williams, Peter (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2021
Description
PF4 (CXCL4) is a cationic platelet chemokine that has been identified as a ligand for the integrin Mac-1 (αMβ2). The interaction between PF4 and Mac-1 has been shown to cause leukocyte migration, improve phagocytosis, and trigger the up-regulation of Mac-1 expression in leukocytes, thereby increasing leukocytic adhesion. Though Mac-1 is known to serve as the site of interaction between PF4 and the leukocyte, the PF4 binding site of Mac-1 remains unknown. 1H-15N HSQC NMR spectroscopy of the interaction between PF4 and Mac-1’s binding site, the αMI domain, can provide this data. This project seeks to create PF4 mutants with site-directed spin labels to enhance the sensitivity of NMR for future experiments that seek to locate the PF4-Mac-1 binding site. It was hypothesized that the mutants created would adopt the native conformation and accept an MTSL label. Two mutants were successfully created and harvested, PF4 S17C and PF4 S26C. Both were soluble and the Sanger sequencing results show that primary structure was conserved except for the substitutions of structurally similar residues indicating the protein folds and likely adopts native conformation. PF4 S26C was labeled with MTSL, and 1H-15N HSQC NMR spectroscopy was performed on unlabeled PF4 S26C (at pH 3.40), MTSL-labeled PF4 S26C (at pH 3.15), and MTSL-labeled PF4 S26C exposed to ascorbic acid (at pH 3.15) to evaluate if the mutant accepts the label and, resultantly, experiences reduced signal intensity. Significant change in signal intensity occurred without change in location of the peaks between the unlabeled and labeled spectra, showing that PF4 S26C accepts the spin label without changing the protein structure and that the label works as expected; however, no change occurred after reducing the spin label with ascorbic acid, preventing confirmation that signal changes were exclusively caused by the MTSL-label. Therefore, though these mutants show potential for future titration with the αMI domain and the hypothesis is supported, a future attempt to reduce MTSL-labeled PF4 S26C at a higher pH (approximately pH 5) is required. Additionally, PF4 S17C should also be evaluated with the methodology used to assess PF4 S26C before its employment in future projects.
ContributorsGamus, Isaac (Author) / Wang, Xu (Thesis director) / Van Horn, Wade (Committee member) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Abstract: It has been established that α-keto-analogs of amino acids can be converted into the amino acids through transamination in vivo. This discovery led to breakthroughs in treating patients who had difficulty digesting traditional proteins, such as in chronic kidney disease (CKD) sufferers where patients have poor kidney function, which poisons the blood with ammonia products.
This pilot study aimed to ascertain the potential for keto acid supplementation in the attempt to supply adequate protein building blocks to healthy populations, with the caveats that said supplementation 1) would utilize non-synthetic methods, 2) offer an alternative to high-phosphate protein supplies such as ruminant animals, and 3) reverse the ill effects of ammonia load by reducing nitrogen intake and consuming ammonia as a fuel for the process of protein synthesis. This proposed solution turns to orange juice and certain varietals of potato juice for their familiarity to consumers, innate nutritional values, and potential for mass-production by many existing companies. The work contained here represents the first phase of experimentation: qualifying the presence of α-keto-analogues of amino acids in these types of produce which, with transamination, could yield the amino acids necessary for adequate protein intake.
Results suggest that these juices do not contain adequate α-keto-analogs of amino acids to supplement proteins in either healthy or ill individuals.
This pilot study aimed to ascertain the potential for keto acid supplementation in the attempt to supply adequate protein building blocks to healthy populations, with the caveats that said supplementation 1) would utilize non-synthetic methods, 2) offer an alternative to high-phosphate protein supplies such as ruminant animals, and 3) reverse the ill effects of ammonia load by reducing nitrogen intake and consuming ammonia as a fuel for the process of protein synthesis. This proposed solution turns to orange juice and certain varietals of potato juice for their familiarity to consumers, innate nutritional values, and potential for mass-production by many existing companies. The work contained here represents the first phase of experimentation: qualifying the presence of α-keto-analogues of amino acids in these types of produce which, with transamination, could yield the amino acids necessary for adequate protein intake.
Results suggest that these juices do not contain adequate α-keto-analogs of amino acids to supplement proteins in either healthy or ill individuals.
ContributorsRex Deltfantan, Kiko (Author) / Wang, Xu (Thesis director) / Maurer, Megan (Committee member) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05