Biochemistry Commons^™

Characterization Of The Function And Regulation Of The Hmpv Phosphoprotein, Rachel Thompson

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus (NNSV) that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. Despite the initial identification of HMPV in 2001, there are currently no FDA approved antivirals or vaccines available. Therefore, understanding the mechanism of HMPV replication is critical for the identification of novel therapeutic targets. A key feature in the replication cycle of HMPV and other NNSVs is the formation of membrane-less, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). Recent work on NNSV IBs suggests they display characteristics of biomolecular condensates formed …

The Development And Characterization Of Nanobodies Specific To Protein Tyrosine Phosphatase 4a3 (Ptp4a3/Prl-3) To Dissect And Target Its Role In Cancer., Caroline Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, …

A Multidisciplinary Characterization Of The Enzymology And Biology Of Reversible Glucan Phosphorylation In Toxoplasma Gondii , Robert Murphy

Theses and Dissertations--Molecular and Cellular Biochemistry

Toxoplasma gondii is an opportunistic, protozoan parasite of all warm-blooded animals, infecting roughly one-third of humans worldwide. Humans acquire infections by consuming T. gondii tissue cysts in undercooked meat or from oocysts shed in cat feces. Encysted parasites convert into rapidly growing tachyzoites that disseminate throughout the body, defining the acute phase of infection. Under host immune pressure, tachyzoites convert into bradyzoites that populate tissue cysts found in CNS or muscle tissue and persist for the lifetime of the host, defining the chronic phase of infection. Tissue cysts are responsible for transmission via carnivory, but also possess the ability to …

Reversible Glucan Phosphorylation In The Red Alga, Cyanidioschyzon Merolae, Corey Owen Brizzee

Theses and Dissertations--Molecular and Cellular Biochemistry

Starch and glycogen are an essential component for the majority of species and have been developed to maintain homeostasis in response to environmental changes. Water-soluble glycogen is an excellent source of quick, short-term energy in response to energy demands. In contrast, plants and algae have developed the macromolecule starch that is elegantly suitable for their dependence on external circumstances. Semi-crystalline starch is water-insoluble and inaccessible to most amylolytic enzymes, thus plants and algae have developed a coordinated system so that these enzymes can gain access to the denser starch energy cache. Starch-like semi-crystalline polysaccharides are also found in red algae, …

Entry And Replication Of Negative-Strand Rna Viruses, Kerri Boggs

Theses and Dissertations--Molecular and Cellular Biochemistry

Hendra virus (HeV) and human metapneumovirus (HMPV) are negative-sense, singled-stranded RNA viruses. The paramyxovirus HeV is classified as a biosafety level 4 pathogen due to its high fatality rate and the lack of a human vaccine or antiviral treatment. HMPV is a widespread pneumovirus that causes respiratory tract infections which are particularly dangerous for young children, immunocompromised individuals, and the elderly. Like HeV, no vaccines or therapies are available to combat HMPV infections. These viruses fuse their lipid envelopes with a cell to initiate infection. Blocking cell entry is a promising approach for antiviral development, and many vaccines are designed …

Substrate Trafficking Within The Type Vii Secretion Systems Of Pathogenic Mycobacteria, Zachary A. Williamson

Theses and Dissertations--Molecular and Cellular Biochemistry

Tuberculosis (TB), primarily caused by infection of Mycobacterium tuberculosis (Mtb) in the lungs, is the deadliest infectious bacterial disease killing 1.5 million people annually. A major determinant of virulence is active secretion through three specialized type VII secretion (ESX) systems; ESX-1, ESX-3, and ESX-5. A large group of substrates exported by the ESX systems is the PE (Proline-Glutamine) and PPE (Proline-Proline-Glutamate) families of proteins, which are highly expanded in the pathogenic species of Mycobacteria and encompass over 7% of Mtb’s genome coding capacity. PE and PPE proteins interact together to form PE-PPE heterodimers, and are secreted through …

Pneumovirus Infections: Understanding Rsv And Hmpv Entry, Replication, And Spread, Jonathan T. Kinder

Theses and Dissertations--Molecular and Cellular Biochemistry

Pneumoviruses including human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are significant causes of respiratory tract infections globally. Children, elderly, and immunocompromised patients are at the greatest risk for developing severe infections, which can have devastating outcomes. Although these viruses are ubiquitous with significant impacts on human health, there are no antivirals or vaccines available. The only FDA approved therapy is a monoclonal antibody for RSV, given prophylactically during the infectious season, and this treatment is only available for high risk infants. The work presented in this thesis aims to increase our understanding of how these viruses enter, replicate, and …

The Oncogenic Role Of The Protein Tyrosine Phosphatase 4a3 (Ptp4a3 Or Prl-3) In T-Cell Acute Lymphoblastic Leukemia, Min Wei

Theses and Dissertations--Molecular and Cellular Biochemistry

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive blood cancer. There are no immunotherapies and few molecularly targeted therapeutics available for the treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression is critical for the development of new therapies for T-ALL. The protein tyrosine phosphatase PTP4A3, also known as PRL-3, has been extensively reported to play a causative role in numerous cancers, including several types of blood malignancies. However, its role in T-ALL is not well defined.

Here, we determined that the PRL-3 plays a critical role in T-ALL initiation and progression by …

Epigenetic Implications In Inorganic Arsenic-Mediated Carcinogenesis, Meredith Eckstein

Theses and Dissertations--Molecular and Cellular Biochemistry

Chronic, low dose exposure to inorganic arsenic (iAs) is a public health concern throughout the world, contributing to the development of many diseases, including lung cancer. Several mechanisms for iAs-mediated carcinogenesis have been proposed, of which the production of reactive oxygen species and formation of chromosomal aberrations are the most studied. Another equally important, yet less studied mechanism is dysregulation of epigenetic marks. “Epigenetics” refers to changes that occur on the DNA and chromatin that do not alter base pair identity, but alter compaction, expression, and regulation of specific DNA sequences. There are several types of epigenetic marks including histone …

The Functional Role Of Rna Binding Protein Rbms3 As A Tumor Promoter In Triple-Negative Breast Cancer Cells, Yuting Zhou

Theses and Dissertations--Molecular and Cellular Biochemistry

RBMS3 belongs to the family of c-myc gene single-strand binding proteins (MSSPs) that play important roles in transcriptional regulation. Here, we show that RBMS3 functions as a tumor promoter in triple-negative breast cancer (TNBC), a highly aggressive BC subtype. Analysis of RBMS3 expression shows that RBMS3 is upregulated at both mRNA and protein levels in TNBC cells. Functionally, overexpression of RBMS3 increases cell migration, invasion and cancer stem cell (CSC) behaviors. Moreover, RBMS3 induces expression of epithelial-mesenchymal transition (EMT) and CSC markers. Conversely, loss of RBMS3 in TNBC BT549 cells inhibits cell proliferation, migration and mesenchymal phenotype. Correlation analysis shows …

Automatic 13C Chemical Shift Reference Correction Of Protein Nmr Spectral Data Using Data Mining And Bayesian Statistical Modeling, Xi Chen

Theses and Dissertations--Molecular and Cellular Biochemistry

Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins. However, due to the intrinsic properties of NMR experiments, results from the NMR instruments require a refencing step before the down-the-line analysis. Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR. There is no available method that can rereference carbon chemical shifts from protein NMR without secondary experimental information such as structure or resonance assignment.

To solve this problem, we …

Computational Tools For The Untargeted Assignment Of Ft-Ms Metabolomics Datasets, Joshua Merritt Mitchell

Theses and Dissertations--Molecular and Cellular Biochemistry

Metabolomics is the study of metabolomes, the sets of metabolites observed in living systems. Metabolism interconverts these metabolites to provide the molecules and energy necessary for life processes. Many disease processes, including cancer, have a significant metabolic component that manifests as differences in what metabolites are present and in what quantities they are produced and utilized. Thus, using metabolomics, differences between metabolomes in disease and non-disease states can be detected and these differences improve our understanding of disease processes at the molecular level. Despite the potential benefits of metabolomics, the comprehensive investigation of metabolomes remains difficult.

A popular analytical technique …

Biochemical Approaches For The Diagnosis And Treatment Of Lafora Disease, Mary Kathryn Brewer

Theses and Dissertations--Molecular and Cellular Biochemistry

Glycogen is the sole carbohydrate storage molecule found in mammalian cells and plays an important role in cellular metabolism in nearly all tissues, including the brain. Defects in glycogen metabolism underlie the glycogen storage diseases (GSDs), genetic disorders with variable clinical phenotypes depending on the mutation type and affected gene(s). Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy and a non-classical GSD. LD typically manifests in adolescence with tonic-clonic seizures, myoclonus, and a rapid, insidious progression. Patients experience increasingly severe and frequent epileptic episodes, loss of speech and muscular control, disinhibited dementia, and severe cognitive decline; death …

Controlling Platelet Secretion To Modulate Hemostasis And Thrombosis, Smita Joshi

Theses and Dissertations--Molecular and Cellular Biochemistry

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent …

Functional Characterization Of Scaffold Protein Shoc2, Hyein Jang

Theses and Dissertations--Molecular and Cellular Biochemistry

Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear.

The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this …

Viral Fusion Protein Tm-Tm Interactions: Modulators Of Protein Function And Potential Antiviral Targets, Stacy Webb

Theses and Dissertations--Molecular and Cellular Biochemistry

Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning …

Platelet Endocytosis: Roles In Hemostasis And Innate Immunity, Meenakshi Banerjee

Theses and Dissertations--Molecular and Cellular Biochemistry

Endocytosis is key to fibrinogen (Fg) uptake, receptor trafficking of integrins (α_IIbβ₃, α_vβ₃) and purinergic receptors (P2Y₁, P2Y₁₂), and thereby for normal platelet function. However, platelet endocytosis could potentially be critical for actively sensing changes in vascular micro-environments and responding accordingly to what is being taken up. This is a more dynamic view of platelets as active surveyors of the vasculature; extending the importance of platelet endocytosis beyond granule biogenesis and perhaps even hemostasis. The mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required …

Investigation Of The Physiological Role Of Rin Gtpase In Cell Death, Axonal Injury, And Inflammation Following Traumatic Brain Injury, Megan Pannell

Theses and Dissertations--Molecular and Cellular Biochemistry

Traumatic brain injury (TBI) is a progressive disorder, in which the primary injury results in the initiation of a complex cascade of secondary biochemical and metabolic changes resulting in lasting neurological dysfunction and cognitive impairment. The heterogeneous nature of the disease has complicated the development of pharmacological agents to improve the outcomes of TBI; to date, no therapeutic treatment has been shown to be effective in clinical trials. Treatments targeting multiple secondary outcomes (cell death, axonal degeneration, and inflammation) may provide enhanced therapeutic efficacy following TBI.

Small Ras family GTP-binding proteins govern diverse cellular processes by directing the relay of …

Rad Gtpase: Identification Of Novel Regulatory Mechanisms And A New Function In Modulation Of Bone Density And Marrow Adiposity, Catherine Nicole Kaminski Withers

Theses and Dissertations--Molecular and Cellular Biochemistry

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not …

Functional Roles For Post-Translational Modifications Of T-Snares In Platelets, Jinchao Zhang

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants …

Calcineurin: From Activation To Inhibition, Erik C. Cook

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin is a Ser/Thr phosphatase whose function is implicated in critical physiological processes such as immune system activation, fetal heart development, and long-term depression in neurons. Calcineurin has been implicated in the progression of Alzheimer’s disease and cardiac hypertrophy. It is not well understood how calcineurin is activated on a molecular level by Ca²⁺ and its activating protein calmodulin. Previous data from our lab show that calmodulin interaction induces the folding of the intrinsically disordered regulatory domain of calcineurin in two discrete and distant regions into α-helical conformations and that this folding is critical for complete activation of calcineurin. …

Physiological Function Of Fus: An Rna Binding Protein In Motor Neuron Disease, Liuqing Yang

Theses and Dissertations--Molecular and Cellular Biochemistry

FUS is an RNA binding protein implicated in the motor neuron disease— amyotrophic lateral sclerosis (ALS, also called Lou Gehrig’s disease). ALS is a fatal neurodegenerative disease characterized by progressive motor neuron death. Mutations in the FUS gene cause about 4% of familial ALS (FUS ALS). Mutated FUS protein mislocalizes from the motor neuron nucleus to the cytoplasm and forms inclusions in the cytoplasm. It is unclear how FUS mislocalization induces motor neuron dysfunction and degeneration. This dissertation research was designed to investigate the physiological functions of FUS in the nucleus, with a purpose to shed light on the pathogenesis …

The Role Of Phlpp In Pancreatic Cancer, Alena J. Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Medicine has come a long way in recent years with reliable treatments for many cancers. Pancreatic ductal adenocarcinoma (PDAC) has very few treatment options available. PDAC has a dismal 5 year survival rate of 4% and a median survival span of 6 months from point of diagnosis; with a high rate of chemotherapy and radiation resistance. A better understanding of the molecular events leading to cancer progression is needed in order to improve the treatment and prognosis of PDAC patients. We begin to elucidate the functional importance of PHLPP on suppressing progression and metastasis of PDAC. PHLPP belongs to a …

Adp-Ribosylation Factor 6 (Arf6) Regulates Integrin Αiibβ3 Trafficking, Platelet Spreading, And Clot Retraction, Yunjie Huang

Theses and Dissertations--Molecular and Cellular Biochemistry

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6^-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6^-/- mice injected with biotinylated-fibrinogen, showed …

Physical Interactions Between Neuropilin And Vegfrs, Integrins In Regulating Endothelial Cell Functions, Xiaobo Li

Theses and Dissertations--Molecular and Cellular Biochemistry

The neuropilin (Nrp) family consists of multifunctional cell surface receptors with critical roles in a number of different cell and tissue types. A core aspect of Nrp function is ligand-dependent cellular adhesion and migration, where it controls the multistep process of cellular motility through integration of ligand binding, receptor coupling and signaling via the coordinated action of its extracellular and intracellular domains. While Nrp regulates cellular adhesion and motility in the cardiovascular and nervous systems under physiological conditions, the emerging pathological role of Nrp in tumor cell migration and metastasis has been identified and provides motivation for continued efforts toward …

Characterization Of Jabba, A Ricin-Resistant Mutant Of Leishmania Donovani, Megan Rhea Phillips

Theses and Dissertations--Molecular and Cellular Biochemistry

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote’s life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one L. donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild-type but similar in size to LPG from wild-type from metacyclic (stationary) phase.

Structural analysis of …

Structural Mechanisms Of Glucan Phosphatase Activity In Starch Metabolism, David A. Meekins

Theses and Dissertations--Molecular and Cellular Biochemistry

Starch is a water-insoluble glucose biopolymer used as an energy cache in plants and is synthesized and degraded in a diurnal cycle. Reversible phosphorylation of starch granules regulates the solubility and, consequentially, the bioavailability of starch glucans to degradative enzymes. Glucan phosphatases release phosphate from starch glucans and their activity is essential to the proper diurnal metabolism of starch. Previously, the structural basis of glucan phosphatase activity was entirely unknown. The work in this dissertation outlines the structural mechanism of activity of two plant glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). The crystal structures of SEX4 …

Molecular Mechanisms Of Neuropilin-Ligand Binding, Matthew W. Parker

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropilin (Nrp) is an essential cell surface receptor with dual functionality in the cardiovascular and nervous systems. The first identified Nrp-ligand family was the Semaphorin-3 (Sema3) family of axon repulsion molecules. Subsequently, Nrp was found to serve as a receptor for the vascular endothelial growth factor (VEGF) family of pro-angiogenic cytokines. In addition to its physiological role, VEGF signaling via Nrp directly contributes to cancer stemness, growth, and metastasis. Thus, the Nrp/VEGF signaling axis is a promising anti-cancer therapeutic target. Interestingly, it has recently been shown that Sema3 and VEGF are functionally opposed to one another, with Sema3 possessing potent …

Neuropilin In The Vascular System: Mechanistic Basis Of Angiogenesis, Hou-Fu Guo

Theses and Dissertations--Molecular and Cellular Biochemistry

The vascular system is critical for maintaining homeostasis in all vertebrates. Structural studies of Neuropilin (Nrp), an essential angiogenic receptor, have defined its role in regulating angiogenesis, the formation of new vessels from the existing vasculature. Utilizing biochemical and biophysical tools we describe the ability of Nrp to function as a co-receptor for the VEGFR receptor tyrosine kinase. Two families of Nrp-1 ligands, Vascular Endothelial Growth Factor A (VEGF-A) and Semaphorin3F (Sema3F), physically compete for binding to the Nrp-1 b1 domain, and have opposite roles. VEGF-A is a potent pro-angiogenic cytokine while Sema3F is an angiogenesis inhibitor. Using coupled structural …

Leptin Resistance Induced Obesity And Diabetes Promote Neuropathological Changes In The Aging Brain, Thomas Platt

Theses and Dissertations--Molecular and Cellular Biochemistry

The aging brain is prone to the development of pathology and dementia. With a rapidly growing elderly population diagnoses of neurodegenerative diseases, such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and Parkinson’s disease are on the rise. Additionally, diabetes and obesity are linked to an increased risk of dementia. The convergence of this increasingly aged population with the obesity and diabetes epidemic give rise to new concerns regarding the future of prevention and treatment of neurodegenerative diseases. Our lab has previously shown that leptin, an adipokine involved in signaling satiety to the hypothalamus, can modulate the generation of the amyloid …