Biochemistry, Biophysics, and Structural Biology Commons^™

Elucidating Functional Roles For Myogenin In Adult Skeletal Muscle Metabolism, Exercise Capacity, And Regeneration, Jesse Flynn

Dissertations and Theses (Open Access)

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox …

Agrobacterium Virb10 Contributions To Type Iv Substrate Secretion, T-Pilus Biogenesis, And Outer Membrane Pore Formation, Isaac Garza

Dissertations and Theses (Open Access)

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) …

Dynamic Remodeling Of The Stressed Heart: Role Of Protein Degradation Pathways, Deborah Vela

Dissertations and Theses (Open Access)

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may …

Understanding Gafp, A Plant Lectin With Broad Spectrum Inhibitory Activity, Alexis Nagel

All Dissertations

South Carolina and Georgia are the largest peach producing regions in the Southeastern United States, generating about $60 million worth (~90,000 tons) of fruit per year on average. Peaches and other stone-fruits (Prunus sp.) can be afflicted by a variety of root-associated diseases which negatively impact annual yield and long-term tree mortality. An engineered Prunus rootstock with enhanced resistance to soil-borne pathogens would therefore be of great benefit to the Southeastern peach industry. The Gastrodia anti-fungal protein (GAFP) is a monocot mannose-binding lectin which is able to inhibit the growth of multiple species of plant pathogenic fungi. Previous findings from …

Spidroin N-Terminal Domain: A Ph Sensor In The Spider Silk Assembly Process, William Gaines

All Dissertations

Spider silks are protein-based fibers with remarkable mechanical qualities. Perhaps even more impressive is the spinning process in which the spider silk proteins (spidroins) are assembled from a highly soluble storage state into a well-ordered and insoluble fiber. Indeed, the ordered arrangement of spidroins, which is endowed by the spinning process, is the basis of fiber strength. However, the forces driving fiber assembly and the mechanisms by which spidroins respond those forces are only poorly understood. Spidroins have a tripartite domain architecture consisting of a large and repetitive central domain flanked by small, non-repetitive N- and C-terminal domains. Both terminal …

Resistance And Dna Repair Mechanisms Under Nitrosative Stress In Mammalian And Microbial Systems, Hyun-Wook Lee

All Dissertations

Living organisms are exposed a nitrosative stress mediated by reactive nitrogen species (RNS) that can cause DNA damage and mutation. DNA base deamination is a typical damage occurred under nitrosative stress, which results in conversion of cytosine (C) to uracil (U), adenine (A) to hypoxanthine (I), and guanine (G) to xanthine (X) or oxanine (O). Base excision repair (BER) is an important pathway to remove deaminated DNA lesions in mammalian and microbial systems. My dissertation work concerns with genes and enzymes involved in resistance to nitrosative stress and DNA glycosylases in the BER pathway. In chapter one, I will briefly …

Applications Of Site-Specific Recombination Systems In Transgene Expression And Marker Gene Removal, Mehmet Aydin Akbudak

Graduate Theses and Dissertations

Site Specific Recombination systems, such as FLP-FRT and Cre-lox, have been successfully used for site-specific gene integration and marker-gene deletion in plant systems. They are very useful tools in the integration of single-copy full-length transgene cassettes into the genome because the transgene integration via conventional methods often generate multi-copy locus. Such complex locus containing direct and inverted repeats of full-length and truncated copies of the transgene cassette generate aberrant RNA resulting in gene silencing. Therefore, for stable gene expression, a single copy transgene locus is preferred. However, even single copy locus sometimes succumbs to gene silencing. Although the mechanism is …

Tryptophan Anchored Peptides In Lipid Bilayer Membranes: Control Of Peptide Orientation And The Phase Behavior Of Cholesterol-Containing Ternary Lipid Mixtures, Johanna Maria Rankenberg

Graduate Theses and Dissertations

Model WALP peptides and "next generation" WALP-derived hydrophobic model peptides were employed to discover principles that govern protein-lipid interactions in biological membranes.

Ternary cholesterol-containing lipid mixtures were examined in the presence of WALP peptides of different lengths (acetyl-GWW(LA)_nLWWA-ethanolamide, with n between 3 and 8). Deuterium NMR spectra from labeled lipids reveal that WALP peptides may stabilize lipid ordered "raft" domains and therefore promote lipid phase separation, albeit to a minor extent. The results depend upon whether dioleolyl- or diphytanoyl-phosphatidylcholine is present as the fluid lipid component.

Several WALP peptides were modified to remove anchoring Trp residues from one …

Intraspecific Variation In Two Cosmopolitan Myxomycetes, Didymium Squamulosum And Didymium Difforme (Physarales: Didymiaceae), Katherine Elizabeth Winsett

Graduate Theses and Dissertations

The myxomycetes (plasmodial slime molds or myxogastrids) are one of three groups considered to be true slime molds (class Eumycetozoa sensu Olive 1975). Two vegetative states--amoebae and plasmodia--along with a spore-producing fruiting body characterize the life cycle of the myxomycetes. These organisms are associated with decaying plant material and are found in all terrestrial habitats worldwide. A number of species are considered cosmopolitan, being found worldwide, where they are associated with a diversity of microhabitats and substrates. A review of the literature, including molecular investigations in all three groups of slime molds, is presented, and this is followed by four …

Kinetic, Mechanistic, And Structural Investigation Of Features Controlling Stereoselectivity Of (R)- And (S)-Hydroxypropyl Com Dehydrogenases From Xanthobacter Autrophicus Strain Py2, Dariusz Adam Sliwa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R …

Requirement For Golgi-Localized Pi(4)P In Fusion Of Copii Vesicles With Golgi Compartments, Andres Lorente-Rodriguez, Charles Barlowe

Dartmouth Scholarship

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) …

Excision Dynamics Of Vibrio Pathogenicity Island-2 From Vibrio Cholerae: Role Of A Recombination Directionality Factor Vefa, Salvador Almagro-Moreno, Michael G. Napolitano, E. Fidelma Boyd

Dartmouth Scholarship

Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. ntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.

An Archaeal Trna-Synthetase Complex That Enhances Aminoacylation Under Extreme Conditions, Vlatka Godinic-Mikulcic, Jelena Jaric, Corinne D. Hausmann, Michael Ibba, Ivana Weygand-Durasevic

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (KD) of 250 nm. Formation of the MtSerRS·MtArgRS complex …

Systematic Approach To Electrostatically Induced 2d Crystallization Of Nanoparticles At Liquid Interfaces, Sumit Kewalramani, Suntao Wang, Yuan Lin, Huong Giang Nguyen, Qian Wang, Masafumi Fukuto, Lin Yang

Faculty Publications

We report an experimental demonstration of a strategy for inducing two-dimensional (2D)crystallization of charged nanoparticles on oppositely charged fluid interfaces. This strategy aims to maximize the interfacial adsorption of nanoparticles, and hence their lateral packing density, by utilizing a combination of weakly charged particles and a high surface charge density on the planar interface. In order to test this approach, we investigated the assembly of cowpea mosaic virus (CPMV) on positively charged lipid monolayers at the aqueous solution surface, by means of in situX-ray scattering measurements at the liquid–vapor interface. Theassembly was studied as a function of the solution …

Work Towards The Isolation And Characterization Of The Muscle Isoform Of Glucose 1,6-Bisphosphatase, Caleb J. Hiller

Theses and Dissertations

Glucose 1,6-bisphosphate is an important small molecule involved in the regulation of glycolysis. Four enzymes synthesize this compound. One enzyme is known to degrade it, glucose 1,6-bisphosphatase. Other groups have produced work that indicates that there are two isoforms of this enzyme, one predominant in the brain and one in the muscle. This thesis contains the work performed in attempts to isolate and characterize the muscle isoform of glucose 1,6-bisphosphatase. While this enzyme was not isolated, much was learned about it and the results from this work may help in the future identification of this enzyme.

Leishmania Major Survival In Selective Phlebotomus Papatasi Sand Fly Vector Requires A Specific Scg-Encoded Lipophosphoglycan Galactosylation Pattern, Deborah E. Dobson, Shaden Kamhawi, Phillip Lawyer, Salvatore J. Turco, Stephen M. Beverley, David L. Sacks

Molecular and Cellular Biochemistry Faculty Publications

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated …

Resonance Assignments And Secondary Structure Predictions Of The As(Iii) Metallochaperone Arsd In Solution, Jun Ye, Yanan He, Jack Skalicky, Barry P. Rosen, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear. We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA. The predicted solution structure of ArsD is in agreement with recently published …

Aminopeptidases Do Not Directly Degrade Tau Protein, K. Martin Chow, Hanjun Guan, Louis B. Hersh

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations.

RESULTS: An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved …

The Mycobacterium Tuberculosis Drugome And Its Polypharmacological Implications, Sarah L. Kinnings, Li Xie, Kingston H. Fung, Richard M. Jackson, Lei Xie, Phillip E. Bourne

Publications and Research

We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today’s most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable …

Dnmt1 Stability Is Regulated By Proteins Coordinating Deubiquitination And Acetylation-Driven Ubiquitination, Zhanwen Du, Jing Song, Yong Wang, Yiqing Zhao, Kishore Guda, Shuming Yang, Hung Ying Kao, Yan Xu, Joseph Willis, Sanford D. Markowitz, David Sedwick, Robert M. Ewing, Zhenghe Wang

Chemistry Faculty Publications

DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation. We describe a previously unknown mode of regulation of DNMT1 protein stability through the coordinated action of an array of DNMT1-associated proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60, which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. In contrast, DNMT1 was stabilized by histone deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus–associated ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as well as the association between HAUSP and DNMT1, suggested that during the cell cycle the initiation …