Biochemistry electronic theses and dissertations (MU)
Permanent URI for this collection
The items in this collection are the theses and dissertations written by students of the Division of Biochemistry. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Columbia. Click on one of the browse buttons above for a complete listing of the works.
Browse
Recent Submissions
Item Structure, mechanism, and inhibition of proline metabolic enzymes(University of Missouri--Columbia, 2025) Meeks, Kaylen; Tanner, John J.Proline metabolic enzymes have been the focus of inhibitor discovery due to their role in cancer cell proliferation and metastasis. The proline biosynthetic enzyme PYCR1 has been associated with high tumor expression, which is linked to poor patient outcomes and tumor aggressiveness. The main focus of this thesis is discovery of novel compounds to inhibit proline biosynthetic and proline catabolic enzymes through structure-based and fragment-based inhibitor discovery methods. These compounds can be used as chemical probes to further elucidate the role of proline metabolism in cancer. Chapter 2 focuses on the optimization of expression and purification of PYCR3, which had previously been insoluble or difficult to purify. Extensive kinetics were performed to determine the mechanism of PYCR3 is most likely the random bi bi mechanism. Additionally, a library of proline analogs was screened against PYCR3. A known PYCR1 inhibitor was found to be 10 times more selective for PYCR1 over PYCR3. A crystallography-based fragment screen performed on PRODH and GSALDH is the focus of Chapter 3. A 288-fragment library was screened in crystallo in cocktails of six fragments. A novel PRODH inhibitor, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site. Analog screening of 4-methoxybenzyl alcohol led to the discovery of a more potent inhibitor of PRODH than the initial hit fragment. Chapter 4 details the screening of 71 fragments against PYCR1 using an enzyme activity assay. Twelve hit compounds were validated with X-ray crystallography. The library was counterscreened against PYCR3 and PRODH. (S)-tetrahydro-2H-pyran-2-carboxylic acid has higher affinity for PYCR1 than NFLP, is 30 times more specific for PYCR1 than PYCR3, and negligibly inhibits PRODH. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate provide the first evidence that the sulfonate group is a suitable replacement for the carboxylate anchor for these enzymes. Chapter 5 serves as a proof-of-concept study for template-based docking on our PYCR1 system. 37 fragment-like carboxylic acids were screened using X-ray crystallography. Strong electron density was observed for 8 compounds, corresponding to a 22% hit rate. The fragments are the first PYCR1 inhibitors to block the P5C and NAD(P)H site simultaneously. This chapter also highlights the importance of crystallography as the primary screening method as only 4 of the 8 hit compounds showed inhibition in enzyme activity assays, highlighting the distinction between binders and inhibitors. 5-oxo-7a-phenyl-hexahydropyrrolo[2,1-b][1,3]thiazole-3-carboxylic acid had a lower IC50 than our benchmark compound NFLP. Combining the success of template-based docking in Chapter 5 and replacement of the carboxylate anchor in Chapter 4, Chapter 6 details a carboxylate bioisosteric library of 22 compounds. Four compounds were found to bind PYCR1 in novel remote binding sites, and inhibit PYCR1 activity, classifying them as allosteric inhibitors. Finally, Chapter 7 contains preliminary data of drug-like molecules screened in a more high throughput approach with enzyme activity assays as the primary screen. 24 compounds appear to inhibit PYCR1 more than NFLP. However, these compounds should be further validated with X-ray crystallography in future studies.Item Development of antibody fragment-based radiotheranostic agents for cancer(University of Missouri--Columbia, 2025) Birikorang, Peggy Afua Agyapomaah; Edwards, BarryWith the increasing clinical use of antigen-targeted therapies for cancer, there is the need for a non-invasive method of assessing target antigen expression prior to treatment. In this study, we report the use of copper-64 (64Cu) radiolabeled anti-EphA2 minibody for pre-treatment diagnosis of antigen expression via Positron Emission Tomography (PET). Minibodies (VH, VL and CH3) are advantageous for use as targeting molecules over intact antibodies (IgG) due to accelerated serum clearance properties while maintaining high uptake and uniform distribution within the tumor, allowing high tumor to background ratios at earlier time points relative to IgG. As EphA2, a tyrosine kinase receptor, is overexpressed in various cancer types with minimal expression in normal tissue, rapid quantification of EphA2 expression could be beneficial for patient stratification. Recombinantly produced anti-EphA2 minibody was evaluated for purity, stability, molecular weight, and binding ability. Following conjugation to the bifunctional chelator, NOTA-SCN, and radiolabeling with 64Cu, [ 64Cu]Cu-NOTA-anti-EphA2 minibody was administered to HT1080-fibrosarcoma tumor-bearing nude mice for in-vivo PET imaging and ex-vivo biodistribution at 4 and 24 hours post-injection (p.i.). The [64Cu]Cu-NOTA-anti-EphA2 minibody exhibited rapid tumor targeting with 25.53 ± 2.92 percent injected dose per gram (%ID/g) at 4 hours, and 22.13 ± 7.68 %ID/g at 24 hours p.i. (n=4). There was fast clearance from the blood, with high tumor to blood ratios. Tumor SUVmean values obtained from region of interest (ROI) quantification of the PET images were 1.13 ± 0.03 and 1.08 ± 0.06 at 4 and 24 hours respectively. These findings demonstrate that anti-EphA2 minibody is an excellent targeting molecule, and [ 64Cu]Cu-NOTA-anti-EphA2 minibody is a promising immunoPET agent with potential for use for other theranostic applications. Targeted radionuclide therapy is rapidly becoming an attractive cancer treatment modality with the recent approval of radiopharmaceuticals like Lutathera and Pluvicto for neuroendocrine tumors and castration-resistant prostate cancers respectively. Despite significant advances made towards their clinical use, there is variability in patient responses, in part attributed to the inter and intra-tumoral heterogeneity of target antigen expression. Furthermore, a fixed dosing regimen, irrespective of tumor burden and physiological properties limits effectiveness of targeted radionuclide therapies. Addressing these limitations to maximize efficacy could significantly enhance the prospects of using targeted radionuclide therapies for cancers such as gliomas, which are resistant to conventional treatment methods. The goal of this project is to test the utility of [64Cu]Cu-NOTA-anti-EphA2 minibody for assessing antigen expression in gliomas for patient stratification, and predicting biological effects of cognate therapeutic isotope, copper-67 (67Cu), to enable more accurate and personalized therapeutic dosing. There is evidence of variable EphA2 expression amongst glioma patients, making it an excellent target for these investigations. The more favorable pharmacokinetics of the 85 kDa minibody compared to whole IgG is leveraged for improved theranostic outcomes. As previously describe, [64Cu]Cu-NOTA-anti-EphA2 minibody was prepared via conjugation of NOTA-SCN to anti-EphA2 minibody and radiolabeling with 64Cu. Orthotopic glioma tumors were established in C57BL/6 mice via the intracranial inoculation of GL261 cells in the right brain hemisphere. About 10 to 14 days post-inoculation, tumor volumes were confirmed via Magnetic Resonance Imaging (MRI), and 11.1 MBq (300 µCi) of [64Cu]Cu-NOTA-anti-EphA2 minibody was administered per mouse for PET/CT imaging at various time points. Image analysis, PET/MRI co-registrations, Standard Uptake Value (SUV) quantifications and dosimetry analysis were performed using Imalytics and OLINDA/EXM. The [64Cu]Cu-NOTA-anti-EphA2 minibody conjugate exhibited high and rapid in-vivo tumor targeting. SUVmean values were 0.66 ± 0.13, 0.73 ± 0.16, 0.88 ± 0.17, 1.18 ± 0.29 and 2.08 ± 0.58 at 1, 4, 10, 24 and 48 hours p.i. respectively. Using voxel-level-based dosimetry analysis via Imalytics, cumulative tumor doses of 0.07 ± 0.02 Gy/MBq and 0.28 ± 0.08 Gy/MBq were computed for [64Cu]Cu-NOTA-anti-EphA2 minibody and its cognate therapeutic counterpart, [67Cu]Cu-NOTA-anti-EphA2 minibody respectively. Effective dose results obtained via Imalytics were consistent with results from OLINDA/EXM, which converts from mouse to human. These results allowed the prediction of dose delivery as well as putative biological effects and/or toxicity of [67Cu]Cu-NOTA-anti-EphA2 minibody on organs like kidneys and heart. In summary, [64Cu]Cu-NOTA-anti-EphA2 minibody is a promising immunoPET agent that could be utilized for cancer diagnosis, antigen expression quantification and dosimetry analysis to guide and inform therapeutic dosing with [67Cu]Cu-NOTA-anti-EphA2 minibody. The cornerstones of targeted radionuclide therapies and other antigen-targeted therapies are effective targeting molecules like antibodies and peptides which play a key role in the precise delivery of cytotoxic payloads to tumor sites by binding to specific tumor-associated antigens or other proteins within the tumor microenvironment. This study focuses on designing, assessing and optimizing the tumor-targeting ability and pharmacokinetics of a novel dual-targeting molecule: anti-EphA2-CD11b bispecific antibody (BsAb). The investigation is geared towards evaluating the potential therapeutic application of this BsAb, which simultaneously targets EphA2, a tumor-associated antigen and CD11b, a protein expressed by immune cells heavily infiltrating the tumor microenvironment. Recombinantly produced anti-EphA2-CD11b-BsAb was conjugated to a bifunctional chelator, NOTA-SCN and then radiolabeled with 64Cu. The [64Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb radioimmunoconjugate was subsequently administered to HT1080-fibrosarcoma-bearing nude mice via tail vein injection. In-vivo PET/CT imaging and ex-vivo biodistribution analysis were performed to determine tumor uptake and pharmacokinetic localization. At 4, 24 and 48 hours p.i., %ID/g values of [64Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb in HT1080 xenograft were 5.35 ± 2.24, 4.44 ± 1.90 and 4.10 ± 0.60 respectively. There was high uptake in the liver as well as CD11b expressing organs: spleen, bone (marrow) and lung. Binding in these CD11b-rich organs were significantly reduced by co-administering the dose with non-radiolabeled anti-CD11b antibody and anti-EphA2- CD11b-BsAb, with the concurrent increase in tumor uptakes compared to non-blocked mice (8.39 ± 1.37 %ID/g for blocked and 4.44 ± 1.90 %ID/g for non-blocked at 24 p.i., P = 0.0175). Further optimization studies showed that at lower molar activity (3.7 MBq/nmol, 100 µCi/nmol), there were significantly higher tumor accumulations and reduced uptakes in CD11b-expressing organs compared to higher molar activity (22.2 MBq/nmol, 600 µCi/nmol). Anti-EphA2-CD11b-BsAb is a functional targeting molecule and would require optimization through molar activity or blocking with non-radiolabeled antibody to maximize tumor targeting.Item Cardiac function and aging in mild and severe mouse models of osteogenesis imperfecta(University of Missouri--Columbia, 2025) Lafaver, Brittany; Phillips, Charlotte L.Osteogenesis imperfecta (OI) is a heritable connective tissue disorder, commonly called brittle bone disease, occurring in approximately 1 in 15,000 births. OI is genetically and clinically heterogeneous with over 1500 known mutations ranging in clinical severity from mild with few fractures and skeletal deformity to severe fragility and perinatal lethality. Mutations in the type I collagen genes, COL1A1 and COL1A2, are present in roughly 85 percent of patients with OI, with other OIcausative mutations arising from gene loci that affect collagen trafficking, processing and post-translational modifications. The term, osteogenesis imperfecta, arises from the imperfect bone formation which results in bone fragility, low bone mineral density and long bone bowing. While bone fragility is the most characterized phenotype in OI, there are many extra-skeletal phenotypes such as skeletal muscle weakness, cardiopulmonary complications, and skin fragility. There is no cure, and current treatment options focus on improving the skeletal complications. OI patients life spans are reported to be reduced by 7-10 years primarily due to cardiopulmonary complications. While pulmonary phenotypes have been investigated in the OI patient community and in multiple mouse models, the cardiac complications have been less studied. As cardiac dysfunction in commonly linked with aging, it is also important to understand the impact of age on heart health in OI. My research sought to better understand the presence of cardiac complications and cardiac consequences of aging in OI by preclinical investigations in the osteogenesis imperfecta murine (oim/oim) model of severe human type III OI. We hypothesized that oim/oim mice would have compromised cardiac function that would worsen with increasing age potentially contributing to shortened life span. To test this hypothesis, we evaluated overall survival to 18 months of age and cardiac function in oim/oim and wildtype mice through in-vivo 7T MRI and echocardiography. We determined that male oim/oim mice have decreased lifespan, with only 50 percent of mice surviving to 18 months, an approximate human age of 56-59 years, and that female oim/oim mice did not exhibit early mortality compared to their wildtype littermates. Additionally, male oim/oim mice exhibited decreased cardiac function and valvular dysfunction at 18 months of age compared to age and sex-matched wildtype littermates. Furthermore, to assess arrhythmogenesis as well as cardiomyocyte health in the oim/oim mouse, isolated hearts and cardiomyocytes were tested for function during stress conditions of preload challenge and increased frequency of contraction, respectively. Our findings demonstrate that oim/oim mice have an increased risk of arrhythmias as a potential contribution of cardiac dysfunction, while isolated oim/oim cardiomyocyte function was equivalent to wildtype cardiomyocyte function. These findings suggest that the type I collagen mutations and the extracellular matrix are likely the driver of cardiac dysfunction in OI and not aberrant cardiomyocyte cellular function. Finally, we also sought to investigate cardiac function in a mild to moderate type I/IV OI mouse model, the +/G610C mouse, with a mild skeletal phenotype. We hypothesized that +/G610C mice would present with cardiac dysfunction at 18 months, but to a lesser degree than the severe oim/oim mouse. To this end, only male +/G610C mice were evaluated, as female mice were not affected in the severe oim/oim model. Survival at 18 months was equivalent in male wildtype and +/G610C mice, and cardiac function appeared to be equivalent at 4 and 18 months of age in male +/G610C mice and age-matched wildtype littermates. These findings suggest that less severe type I collagen variants may have reduced risks for development of cardiac dysfunction. However, a limitation of this study is that wildtype laboratory mice have an average lifespan of 28 months, and 18 months of age may have been insufficient to characterize cardiac function and aging in the +/G610C mouse. Overall, my research was the first to show altered lifespan in the preclinical oim/oim mouse model, with the cardiac manifestations as a potential contributor to early mortality. I am also the first to show age, sex and severity dependent phenotypes in the heart, indicating that further studies are needed to fully elucidate the role of type I collagen defects in heart health in OI. My work revealed that at the cellular level cardiomyocyte health does not appear compromised, suggesting that type I collagen mutations primarily compromise the extracellular matrix and not cardiomyocyte function during stress. Oim/oim mouse hearts exhibited an increased risk for developing arrythmias as compared to wildtype hearts. This body of work contributes to a growing awareness of the extra skeletal manifestations of OI as a type I collagenopathy in aging. Future natural history studies that evaluate aging in a temporal manner, as well as mechanistic studies of cardiac health in OI remain to be pursued, these novel findings emphasize the need for further cardiovascular research and careful cardiac monitoring for the OI patient population and contributes to the overall knowledge of type I collagen's role in cardiac health.Item Novel roles of dynamin-related protein in iron-deficiency in Arabidopsis thaliana(University of Missouri--Columbia, 2024) Antoine-Mitchell, Alani; Heese, AntjePlants have developed sophisticated mechanisms to maintain optimal homeostasis for their survival in diverse environmental conditions. Among these mechanisms, the modulation of the plasma membrane (PM) proteome stands out as a crucial adaptation strategy. The PM serves as a dynamic interface essential for perceiving and adapting to extra- and intracellular stimuli, necessitating precise control over the composition of its proteome. One such control that is central to the fine-tuning of the PM protein composition is achieved through vesicular trafficking. Vesicular trafficking encompasses the sorting and trafficking of cargo proteins to and from the PM via the central intracellular sorting hub trans-Golgi network/early endosomes (TGN/EE). However, the roles of components involved in the post-Golgi trafficking of proteins required for critical cellular processes remain largely unknown. Here, we utilized the model plant Arabidopsis thaliana (Arabidopsis) as well as biochemical and transcriptomic approaches to uncover novel roles of the vesicular trafficking protein DYNAMIN-RELATED PROTEIN1A (DRP1A) in modulating the accumulation of proteins and cellular components essential for physiological processes including nutrient deficiency. In Arabidopsis, DRP1A is a plant-specific large GTPase that belongs to a family of sixteen dynamin-related proteins (DRPs). DRPs are divided into six subfamilies (DRP1-6) based on their domain structure and functions in fission of organelles and/or membrane vesicles. DRP1A is the best-studied of the five DRP1s (DRP1A-E) and studies have shown that loss-of-function drp1a mutants are defective in cytokinesis and constitutive bulk membrane endocytosis. Previous studies from our lab identified DRP1A in modulating the proper PM abundance of the leaf-specific immune receptor FLAGELLIN SENSING2 (FLS2) after elicitation with the bacterial pathogen-associated molecular pattern (PAMP) flg22. Underscoring its role as an endocytic accessory protein, drp1a mutants are impaired in the flg22-induced endocytosis of FLS2. DRP1A is also essential for the polar PM localization of root-specific hormone efflux carrier PINFORMED1 (PIN1) and the nutrient transporter BORON TRANSPORTER1 (BOR1) to specific subdomains of the PM. Given the significance of DRP1A in maintaining abundance and polar localization of PM proteins, we sought to identify other PMlocalized proteins that may be modulated by DRP1A. Vesicular trafficking regulates the PM abundance and polar localization of the root-specific iron (Fe) transporter IRON REGULATED TRANSPORTER1 (IRT1). While few vesicular trafficking components have been identified in this process, this study identified a novel role for DRP1A in modulating the abundance of IRT1 protein and mRNA under various growth conditions. We discovered that loss of DRP1A led to reduced PM accumulation of IRT1 which correlated with reduced Fe in root tissue of drp1a mutant seedlings as well as reduced chlorophyll content and protein accumulation of photosynthetic proteins that require Fe for their function and/or stability. Conversely, we uncovered that drp1a mutants showed increased protein accumulation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), potentially leading to increased intracellular reactive oxygen species (ROS). In turn, increased intracellular ROS may correlate with disrupting Fe-deficiency pathways, indicating a role for DRP1A in Fe homeostasis and stress responses. Under Fe-deficiency, IRT1 must be trafficked and reside in the soil-side of the PM, called outer domain, for uptake of Fe from the rhizosphere as needed; but IRT1 must be removed from the PM through endocytic internalization to prevent excess Fe uptake, which can be toxic. When exploring the physiological impact of loss of DRP1A during Fe-deficiency, we uncovered that under both continuous Fe-deficiency and induced Fedeficiency, drp1a mutants had reduced PM accumulation of IRT1 as well as reduced accumulation of PHOTOSYNTHETIC ELECTRON TRANSFER C, a photosynthetic protein that requires Fe as part of its Fe-S cluster to transport electrons from Photosystem II to Photosystem I. Additionally, we provide evidence that DRP1A played a role in modulating the PM abundance of RBOHD that appeared to be independent of induced Fe-deficiency. Taken these findings together, this study provides new insight on the role of DRP1A in modulating Fe-deficiency responses in plants, though much remains unknown about the underlying cellular and molecular mechanisms. Lastly, through qRT-PCR and RNA-seq analysis, we discovered that loss of DRP1A altered the transcriptional response of a subset of Fe-response genes, specifically the major Fe-deficiency transcription factor FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) and its downstream targets IRT1 and FERRIC REDUCTASE OXIDASE2 (FRO2). Our findings revealed DRP1A's specific role in potentially modulating the transcriptional response to Fe-deficiency in roots at the level of FIT during induced Fe-deficiency. These genes are root-specific Fe-response genes and leaf-specific Fe-response genes such as OLIGOPEPTIDE TRANSPORTER3 (OPT3) and POPEYE (PYE) were not significantly altered in the leaf, we potentially identified a role for DRP1A in propagating the yet unknown Fe-deficiency signal within the roots, leading to reduced FIT gene expression. These findings highlight an essential role of DRP1A in the modulating the abundance of membrane proteins and transcriptional regulation of a subset of genes many of which have roles in maintaining Fe-homeostasis. With increasing global food demands, understanding the role of vesicular trafficking in Fe uptake and utilization is critical for developing sustainable, nutrient-rich crops to combat malnutrition and food insecurity.Item Structural studies of domain of unknown function 507 (DUF507)(University of Missouri--Columbia, 2023) McKay, Cole Ethan; Tanner, John J.The crystal structure of the domain of unknown function family 507 protein from Aquifex aeolicus is reported (AaDUF507, UniProt O67633, 183 residues). The structure was determined in two space groups (C2221 and P3221) at 1.9 A resolution. The phase problem was solved by molecular replacement using an AlphaFold model as the search model. AaDUF507 is a Y-shaped a-helical protein consisting of an anti- parallel 4-helix bundle base and two helical arms that extend 30-A from base. The two crystal structures differ by a 25 degrees rigid body rotation of the C-terminal arm. The tertiary structure exhibits pseudo-twofold symmetry. The structural symmetry mirrors internal sequence similarity: residues 11-57 and 102-148 are 30 percent identical and 53 percent similar with an E-value of 0.002. In one of the structures, electron density for an unknown ligand, consistent with nicotinamide or similar molecule, may indicate a functional site. Docking calculations suggest potential ligand binding hot spots in the region between the helical arms. Structure- based query of the Protein Data Bank revealed no other protein with a similar tertiary structure, leading us to propose that AaDUF507 represents a new protein fold.