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Description
Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation of S-cysteinylated albumin in P/S samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. To emphasize the capability of this assay, a blind challenge demonstrated the ability of ΔS-Cys-Albumin to detect exposure of individual and grouped P/S samples to unfavorable storage conditions. The assay was also capable of detecting an anomaly in a case study of nominally pristine serum samples collected under NIH-sponsorship, demonstrating that empirical evidence is required to guarantee accurate knowledge of archived P/S biospecimen storage history.
The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
ContributorsJeffs, Joshua W (Author) / Borges, Chad R (Thesis advisor) / Van Horn, Wade (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2018
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
Plasma and serum are the most commonly used liquid biospecimens in biomarker research. These samples may be subjected to several pre-analytical variables (PAVs) during collection, processing and storage. Exposure to thawed conditions (temperatures above -30 °C) is a PAV that is hard to control, and track and could provide misleading information, that fail to accurately reveal the in vivo biological reality, when unaccounted for. Hence, assays that can empirically check the integrity of plasma and serum samples are crucial. As a solution to this issue, an assay titled ΔS-Cys-Albumin was developed and validated. The reference range of ΔS-Cys-Albumin in cardio vascular patients was determined and the change in ΔS-Cys-Albumin values in different samples over time course incubations at room temperature, 4 °C and -20 °C were evaluated. In blind challenges, this assay proved to be successful in identifying improperly stored samples individually and as groups. Then, the correlation between the instability of several clinically important proteins in plasma from healthy and cancer patients at room temperature, 4 °C and -20 °C was assessed. Results showed a linear inverse relationship between the percentage of proteins destabilized and ΔS-Cys-Albumin regardless of the specific time or temperature of exposure, proving ΔS-Cys-Albumin as an effective surrogate marker to track the stability of clinically relevant analytes in plasma. The stability of oxidized LDL in serum at different temperatures was assessed in serum samples and it stayed stable at all temperatures evaluated. The ΔS-Cys-Albumin requires the use of an LC-ESI-MS instrument which limits its availability to most clinical research laboratories. To overcome this hurdle, an absorbance-based assay that can be measured using a plate reader was developed as an alternative to the ΔS-Cys-Albumin assay. Assay development and analytical validation procedures are reported herein. After that, the range of absorbance in plasma and serum from control and cancer patients were determined and the change in absorbance over a time course incubation at room temperature, 4 °C and -20 °C was assessed. The results showed that the absorbance assay would act as a good alternative to the ΔS-Cys-Albumin assay.
ContributorsJehanathan, Nilojan (Author) / Borges, Chad (Thesis advisor) / Guo, Jia (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2022
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
This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
ContributorsNagaratnam, Nirupa (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually high number of predicted outer surface lipoproteins of unknown function but with multiple complex roles in pathogenesis, and an unusually low number of predicted outer membrane proteins, given the necessity of bringing in the required nutrients for pathogen survival. Cellular processing of bacterial membrane proteins is complex, and structures of proteins from Bb have all been solved without the N-terminal signal sequence that directs the protein to proper folding and placement in the membrane. This dissertation presents the first membrane-directed expression in E. coli of several Bb proteins involved in the pathogenesis of Lyme disease. For the first time, I present evidence that the predicted lipoprotein, BBA57, forms a large alpha-helical homo-multimeric complex in the OM, is soluble in several detergents, and purifiable. The purified BBA57 complex forms homogeneous, 10 nm-diameter particles, visible by negative stain electron microscopy. Two-dimensional class averages from negative stain images reveal the first low-resolution particle views, comprised of a ring of subunits with a plug on top, possibly forming a porin or channel. These results provide the first evidence to support our theories that some of the predicted lipoproteins in Bb form integral-complexes in the outer membrane, and require proper membrane integration to form functional proteins.
ContributorsRobertson, Karie (Author) / Hansen, Debra T. (Thesis advisor) / Fromme, Petra (Thesis advisor) / Van Horn, Wade (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
In this study, the stability of two protein homo-oligomers, the β clamp (homodimer) from E. coli and the Proliferation Cell Nuclear Antigen (PCNA) from the yeast cell, were characterized. These clamps open through one interface by another protein called clamp loader, which helps it to encircle the DNA template strand. The β clamp protein binds with DNA polymerase and helps it to slide through the template strand and prevents its dissociation from the template strand. The questions need to be to answered in this research are, whether subunit stoichiometry contributes to the stability of the clamp proteins and how does the clamp loader open up the clamp, does it have to exert force on the clamp or does it take advantage of the dynamic behavior of the interface?
The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.
In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.
In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
ContributorsPUROHIT, ANIRBAN (Author) / Levitus, Marcia (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Yan (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