Biochemistry electronic theses and dissertations (MU)
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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.
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Item Therapeutic targeting of the ATP7A copper transporter in cancer(University of Missouri--Columbia, 2025) Azubuogu, Chiemerie Chibuzor; Petris, Michael J.[EMBARGOED UNTIL 12/01/2026] Copper (Cu) is an essential micronutrient involved in numerous biochemical processes, including energy production, iron metabolism, cell proliferation, cuproenzyme activity, and collagen crosslinking. Because Cu cannot be synthesized or metabolized by the body, it must be obtained through diet or supplementation. As a redox-active metal, Cu cycles between cuprous (Cu⁺) and cupric (Cu²⁺) states, making it a critical cofactor for various cuproenzymes. However, this same redox activity renders free Cu ions highly cytotoxic within the cytosol. To mitigate this, cells have evolved to control Cu uptake, intracellular distribution, and export. The high-affinity Cu transporter 1 (CTR1) mediates cellular Cu uptake, while the P1B-type ATPase ATP7A facilitates Cu transport from the cytosol into the lumen of vesicles or the trans-Golgi network (TGN), where it metalates cuproenzymes such as the lysyl oxidase (LOX) family and tyrosinase. Under elevated Cu conditions, ATP7A undergoes Cu-stimulated trafficking to the plasma membrane to export excess Cu into the extracellular space. Studies have shown that many cancer cells exhibit increased Cu demand. Elevated Cu levels have been linked to enhanced kinase activity of MEK1/2 and ULK1/2, promoting tumor growth in BRAF- and KRAS-driven cancers. Cu has also been reported to induce unfolding of the tumor suppressor protein p53 and to drive invasion and chemoresistance in various cancer types. The heightened metabolic demand for Cu in cancer cells is a vulnerability that can be explored in cancer-targeted therapeutic solutions. Several studies have demonstrated that Cu chelation using agents such as penicillamine, trientine, tetrathiomolybdate, and tetraethylenepentamine, significantly reduces primary tumor growth, metastasis, and angiogenesis in preclinical cancer models. However, clinical trials using these chelators have yet to yield viable cancer therapies. The first part of this dissertation explores an alternative systemic Cu depletion strategy by targeting intestinal Cu absorption through deletion of the Atp7a gene. We demonstrate that intestinal Atp7a deletion in C57BL/6 mice, eliminates ATP7A protein expression in the small intestine, induces systemic Cu deficiency, and reduces Cu levels in both LLC and B16 primary tumors. Furthermore, we show that this targeted Atp7a deletion enhances the therapeutic efficacy of the Cu chelator tetrathiomolybdate in suppressing tumor growth in both B16 and LLC tumor models, and spontaneous metastasis in the LLC tumor model. In addition to its role in Cu homeostasis, ATP7A is implicated in the development of chemoresistance in various cancers. Deletion of Atp7a in RAS-transformed mouse embryonic fibroblasts (MEFs) increases sensitivity to cisplatin in vivo. ATP7A and its homolog ATP7B are thought to confer chemoresistance by sequestering drugs in vesicles or the TGN, although the precise mechanisms remain under debate. The second part of this dissertation investigates the roles of ATP7A functions in the development of chemoresistance in 4T1 TNBC cells and the underlying mechanisms. Using a novel small-molecule ATP7A inhibitor, MKV3, we show that ATP7A inhibition significantly reduces cellular GSH levels, enhances doxorubicin (DOX) sensitivity, and reverses DOX resistance. Additionally, ATP7A inhibition increases nuclear DOX accumulation, reduces DOX efflux, and promotes DOX-induced aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT) proteins in 4T1 TNBC cells. Altogether, these studies support ATP7A as a promising therapeutic target for both cancer treatment and the reversal of acquired chemoresistance.Item Structural dynamics to therapeutic interactions : visualizing proline metabolic enzymes by kinetic crystallography and molecular dynamics, and antibody-antigen complexes from enterotoxigenic E. coli by cryo-EM(University of Missouri--Columbia, 2025) Buckley, David Pryor; Tanner, John J.; Berndsen, Zachary T.Structural studies using molecular dynamics simulations, X-ray crystallography, negative stain and cryo-electron microscopy on a diverse set of disease-related contexts (i.e., cancer metastasis, proline metabolic disorders, and enterotoxin-induced diarrheal illness) are presented in this thesis. Insights from the dynamics and structural descriptions obtained from this work contribute essential knowledge for the development of treatments to these diseases. Chapter 1 describes a collaborative project initiated by Dr. Marie Migaud, at the University of South Alabama, an expert in synthetic organic chemistry and disease involving aberrant forms of metabolic dinucleotides. We show via molecular dynamics (MD) simulations that three ROS-generated oxidative states on the nicotinamide of NAD(H), termed ox-NADs, display distinct conformational preferences in solution, with comparisons drawn to the solution dynamics of the essential dinucleotides NAD+ and NADH. The mechanism of exactly how ox-NADs contribute to their observed role in cancer metastasis and age-related diseases is unknown. Current hypotheses are either (1) given their high similarity to NAD(H), they could behave as endogenous inhibitors to NAD(H)-binding proteins, or (2) they exacerbate disease progression via the depletion of available NAD(H) in the cell. Future work to test the first hypothesis could involve obtaining X-ray crystal structures of ox-NADs (primarily 4-ox-NAD, which behaved most similar to NAD(H) from our analyses) bound to NAD(H)-binding proteins, in addition to thorough binding assays. Chapter 2 details work centered around a medical case study through collaboration with medical researchers at the United Hospitals of Marche, Italy, in which a young child displayed symptoms of pyridoxine dependent epilepsy (PDE) linked to two missense mutations in their ALDH7A1 gene. When I joined the project, one of these point mutations in the ALDH7A1 protein, R134S, was well characterized structurally, and the biochemical reasoning for the reduced activity of the mutated ALDH7A1 that leads to PDE was clear (i.e., disruption of the tetrameric oligomer required for ALDH7A1 catalysis). The other mutation, R441C, displayed very similar structural characteristics to wildtype ALDH7A1, thus an explanation for the drastic 50-fold reduction in catalytic activity could not be drawn. I ran a series of MD simulations of the structure of the mutant R441C ALDH7A1 tetramer, along with simulations of the wildtype ALDH7A1 tetramer, and identified a very subtle, but apparent difference in the dynamics of the C-terminal “gate” that has been previously shown in ALDH7A1 to have a significant function in modulating substrate access to its active site. This work neatly combines X-ray crystallography, enzyme kinetics, sedimentation velocity analysis, and MD simulations to deduce the mechanisms of the two identified mutations in their contribution to PDE in the young child of this case study. Chapter 3 surrounds my most substantial, primary project under Dr. Tanner using X-ray crystallography and MD to study substrate channeling in the bifunctional proline catabolic enzyme, Sinorhizobium meliloti Proline Utilization A (SmPutA). The human forms of the individual, monofunctional enzymes PRODH and ALDH4A1 (a.k.a. GSALDH) that achieve this essential metabolic conversion of L-proline to L-glutamate are thought to engage in a protein-protein interaction and undergo substrate channeling similar to bacterial PutAs, and both enzymes have substantial implications in proline metabolic disorders, cancer, and atherosclerosis. Utilizing kinetic X-ray crystallography, or in crystallo enzymatic turnover of SmPutA, transient covalent intermediates and substrate/product complexes were captured, which provides a near complete structural understanding of how proline catabolism is achieved in PutA. We initiated MD simulations from one of the captured species, the PRODH product P5C-bound state, and observed product release events in the majority of simulations and sequestering of this intermediate within the large tunnel that connects the PRODH and ALDH4A1 active sites. This work provides the first ever MD simulations of any PutA, and could inform on how this process is achieved in humans (specifically in our description of the absolute requirement of the conserved aspartate-arginine ion gate in the PRODH active site opening prior to product release). Chapter 4 highlights my work after formal addition of Dr. Berndsen as my co-advisor, where we collaborated with Dr. James Fleckenstein at Washington University in St. Louis, a leader in the field of the molecular pathogenesis and prevention of enterotoxigenic E. coli (ETEC). ETEC is among the leading causes of diarrheal disease in young children in low-middle income countries, in regions lacking access to the fundamental need of clean drinking water. In this chapter, I contributed extensive negative stain electron microscopy data that outlined differential polyclonal antibody (pAb) targeting against a virulence factor and adhesin of ETEC, EtpA, from the sera of mice vaccinated with EtpA adjuvanted with double mutant heat-labile enterotoxin (dmLT; LT is also secreted by ETEC, one of the direct causative toxins of eventual diarrhea, and an activator of innate immunity in its double mutant form). We were able to obtain 2-dimensional class averages which detail a time-dependence during vaccination on the quality and quantity of pAbs targeting EtpA. EtpA is a ripe target in vaccine design due to its role in facilitating ETEC colonization, and this work aids in development of an ETEC vaccination strategy using EtpA. Chapter 5 describes my most substantial, primary project under Dr. Berndsen, and continued collaboration with the Fleckenstein lab, where we focus on another secreted virulence factor of ETEC, EatA (a serine protease autotransporter, or SPATE), using cryo-electron microscopy (cryo-EM). The role of EatA during ETEC infection is in its mucinase activity, specifically in cleaving the layer of complex glycoprotein MUC2 that lines intestinal epithelia. The Fleckenstein lab has identified human antibodies against EatA that neutralize MUC2 degradation through a combination of natural ETEC infection cases and controlled ETEC infection studies. We have obtained high resolution structures of four of the identified antibodies in their fragment antigen binding (Fab) form bound to EatA, and detail three unique EatA epitopes. We also show that one of the targeted epitopes is highly conserved in the related Shigella and enteroaggregative E. coli SPATE molecules, SepA and Pic, by obtaining cryo-EM Fab complexes with these molecules that display near identical binding modes to the Fab-EatA solved complex. Together, these results promote this identified structural motif as a prime candidate for design of a minimal immunogenic construct in the effort to create a broadly protective vaccine against ETEC and related enteric pathogens. Chapters 1-4 are adapted from published works, and chapter 5 is currently in the final stages of manuscript preparation for initial submission to bioRxiv.Item Structural and biochemical characterization of soybean serine hydroxymethyltransferase(University of Missouri--Columbia, 2025) Owuocha, Luckio Frank; Beamer, Lesa J.[EMBARGOED UNTIL 08/01/2026] Serine hydroxymethytransferase (SHMT) is a pyridoxal 5-phosphate dependent enzyme that is critical in one carbon metabolism. SHMT catalyzes the conversion of serine and tetrahydrofolate (THF), to glycine and 5,10 methylene-THF feeding one carbon to folate dependent processes like purine, thymidylate and methionine biosynthesis which are vital to cellular growth and proliferation. SHMT is therefore, increasingly a target of interest in the design of chemotherapeutic, antimalarial, antimicrobial and herbicidal agents. SHMT has also been implicated in soybean's resistance to soybean cyst nematode. Two polymorphisms (P130R and N358Y) in SHMT8 (cytosolic enzyme), found in the resistant soybean cultivar Forrest, impair enzyme activity by affecting the folate-binding loop. Since soybean cyst nematode (SCN) depends on soybean-derived folate, this reduced activity may contribute to SCN resistance by disrupting folate metabolism. Soybean SHMT presents a unique research opportunity, because it is easily crystallizable and has several established biochemical characterization protocols. Since SHMT is a ubiquitous enzyme, structural and even biochemical data may be extrapolated to other kingdoms of life. In this work we have characterized structurally through X-ray crystallography and biochemistry, polyglutamylated-THF, the second cytosolic enzyme SHMT5 and two natural variants I37F and N358H. The high-resolution structure of SHMT8 complexed with polyglutamylated-THF provides key insights into polyglutamylated-THF interactions and may serve as a valuable structural template for future molecular modeling and antifolate inhibitor design. Structural and biochemical findings from SHMT5, I37F, and N358H provide deeper insight into folate metabolism and its role in SCN resistance, offering a foundation for potential crop improvement strategies.Item Prenatal and longitudinal study of myostatin inhibition on maternal and offspring muscle and bone health in osteogenesis imperfecta mice(University of Missouri--Columbia, 2025) Crawford, Tara Kayann; Phillips, Charlotte L.[EMBARGOED UNTIL 08/01/2026] Osteogenesis imperfecta (OI) is a rare heritable connective tissue disorder most often caused by mutations in the type I collagen genes, COL1A1 and COL1A2, and affects approximately 1:15,000 live births. The hallmark of OI is skeletal fragility, however due to the abundance of type I collagen found in many tissues and organ systems, other manifestations of OI include skeletal muscle weakness, cardiopulmonary complications, and even early death. Skeletal muscle weakness, now known to be intrinsic to OI, affects approximately 80% of patients as well as the osteogenesis imperfecta murine (oim) mouse model. There is no cure for OI and treatment strategies have focused mainly on mitigation of fractures through anti-resorptive therapy with limited success. The recent recognition of OI as a type I collagenopathy and its extensive genetic and clinical heterogeneity provide even further challenges for the identification of effective therapeutic strategies. Recent improvements in prenatal screening have advanced the identification of OI in utero, however currently employed treatment techniques are not compatible with gestation, lactation, or early postnatal care for maternal or child health. Previous research highlights the critical need for novel therapeutics to combat bone related loss during pregnancy and lactation, which can be particularly detrimental to OI patients. While direct bone alterations remain the forefront of most therapeutic avenues, bone may also be altered via muscle through muscle-bone crosstalk. One robust therapeutic target for muscle has been myostatin, a negative regulator of muscle growth. Many studies investigating myostatin deficiency, as well as pharmacological inhibition of myostatin have reported evidence of increased skeletal muscle mass and subsequent bone heath. The goal of this dissertation was to investigate the health and safety, as well as the efficacy of utilizing a humanized monoclonal anti-myostatin antibody (Mstn-Ab) therapy during the preconception, prenatal, and early postnatal time periods in the oim mouse model to enhance maternal and offspring musculoskeletal health. A first step was to confirm the compatibility of Mstn-Ab therapy with maternal health, pregnancy, and fetal development. Towards this end, we first examined the impact of maternal Mstn-Ab treatment during the prenatal period alone in wildtype (WT) and heterozygous osteogenesis imperfecta murine (+/oim) female mice at two critical timepoints, late gestation and following lactation. Non-pregnant and pregnant prenatally treated mice and their fetuses were examined at embryonic day E17.5 (late gestation). We also examined prenatally treated dams following lactation at postpartum day P21 and monitored their offspring to evaluate the impact of prenatal exposure to Mstn-Ab therapy into adulthood (16 weeks, the age of peak bone mass). We demonstrated that Mstn-Ab therapy was safe for both maternal and fetal tissues as determined by pregnancy outcomes including metabolic and muscle health, and skeletal mass and integrity. However, we also determined that prenatal exposure to Mstn-Ab therapy alone was not sufficient to improve maternal muscle weakness or prevent bone loss due to pregnancy and lactation. Having established the health and safety of Mstn-Ab in utero, we then examined longterm Mstn-Ab treatment in WT and +/oim dams administered during the preconception, prenatal, perinatal, and lactation time periods (to be evaluated following lactation), as well as continued postnatal treatment in offspring into adulthood (to be evaluated at 16 weeks of age). To further assess the level of effectiveness of Mstn-Ab therapy, untreated WT and myostatin deficient (Mstn-het) dams and offspring were also evaluated. Our findings demonstrate that long-term maternal administration of Mstn-Ab treatmentinitiated preconception and followed through lactation improved maternal muscle mass with some increases in bone microarchitecture compared to Ctrl-Ab treated dams. This increase in musculoskeletal outcomes was able to mimic those seen in untreated Mstn-het dams, suggesting that preconception through lactation is a sufficient treatment window of Mstn-Ab to model positive musculoskeletal health of myostatin deficiency. Furthermore, longitudinal treatment of Mstn-Ab continued in offspring after weaning improved offspring musculoskeletal outcomes, even in the severe oim/oim offspring cohorts compared to their Ctrl-Ab treated counterparts. Overall, this research sought to identify a safe and effective therapeutic in the oim mouse model of OI that was compatible with gestation and early postnatal care, as well as aid in maternal health during pregnancy and lactation. This study is the first to demonstrate evidence of significant and potentially detrimental changes that occur in bone microarchitecture, biomechanical, and biochemical properties during pregnancy and lactation in a mouse model of OI. We further demonstrated that lactation related bone loss can be reduced through pharmacological inhibition of myostatin via humanized monoclonal anti-myostatin antibodies administered from preconception through lactation. This treatment also was effective when maintained in offspring post-weaning through adulthood to improve musculoskeletal health, even in severe models of OI.Item Investigating the role of plasma membrane E3 ligase, ATL6, and the CBL-CIPK signaling network as novel regulators of pattern triggered immunity in plants(University of Missouri--Columbia, 2025) Alcantar, Martin; Peck, Scott C.[EMBARGOED UNTIL 08/01/2026] When a plant and potential pathogen interact, a race ensues to deploy their respective defense and virulence mechanisms. Pattern triggered immunity (PTI), a plant's first line of defense, is initiated when surface localized pattern recognition receptors (PRRs) recognize evolutionarily conserved molecules known as pathogen associated molecular patterns (PAMPs) from the invading pathogen. Initiation of PTI is characterized by a variety of intracellular molecular responses including activation of the MAPK cascade, calcium influx, and regulation of the abundance of bioactive metabolites in the apoplast. The goal of some or all of these responses is to stop pathogen growth. Concurrently, when a bacterial pathogen recognizes the plant via plant-derived metabolites, it activates its Type III Secretion System (T3SS) to inject effector proteins into the plant cell to suppress host defense responses. If the pathogen manages to inject effectors and suppress defense, then PTI fails. Therefore, PTI must occur rapidly to prevent the bacteria from reaching this virulent stage. Previous work from our lab identified plants lacking the negative regulator of defense, MAP KINASE PHOSPHATASE 1 (MKP1), that are enhanced in their ability to defend against the bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000). mkp1 plants restrict effector delivery into the plant resulting in greater resistance than in wild-type plants. This resistance is due to a decrease in the abundance of extracellular T3SS-inducing metabolites compared to wild-type plants. A key question remains as to how the plant regulates the accumulation of the T3SS-inducing metabolites. The work in this dissertation establishes the PM-localized E3 ligase, ATL6, as a novel positive regulator required for PTI-associated resistance, while having no apparent impact on other canonical PTI molecular responses. Using an mkp1 atl6 double mutant, I have established that ATL6 plays an epistatic role to MKP1 in conferring enhanced resistance. I have also identified that ATL6 is necessary for suppression of the bacterial T3SS, suggesting a role in regulation of accumulation of the T3SS-inducing metabolites. Because ATL6 is phosphorylated during PTI, I aimed to identify the kinase(s) responsible for this activation. From the phosphorylation motif, I have identified a family of calcium sensing proteins and their interacting protein kinases (CBL-CIPK) that may be responsible for activation of ATL6. I have demonstrated that eliminating all PM-localized CBLs, which in turn eliminates all CIPK activity at the plasma membrane (where ATL6 is located) results in plants that are unable to establish PTI. Furthermore, I have shown that knocking out a subset of PM-localized CBLs is enough to eliminate mkp1-mediated enhanced resistance. This work identifies a new role for ATL6 in establishing PTI, likely through regulation of T3SS-activating metabolite abundance resulting in restriction of T3SS- activation. In addition, this work has identified new signaling components (CBL-CIPKs), required for full PTI-induction, bringing us one step closer to completing the major signaling pathway responsible for PTI.