Biochemistry Commons^™

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 …

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 …

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 …

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. …

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 …

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 …

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Investigating Therapeutic Options For Lafora Disease Using Structural Biology And Translational Methods, Amanda R. Sherwood

Theses and Dissertations--Molecular and Cellular Biochemistry

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as …

Structural Basis Of Substrate Recognition In Thimet Oligopeptidase And Development Of Nanoparticles For Therapeutic Enzyme Delivery, Jonathan Mark Wagner

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes …