Biochemistry Commons^™

Mimosa: A System For Minimotif Annotation, Jay Vyas, Ronald J. Nowling, Thomas Meusburger, David P. Sargeant, Krishna Kadaveru, Michael R. Gryk, Vamsi Kundeti, Sanguthevar Rajasekaran, Martin Schiller

Life Sciences Faculty Research

BACKGROUND:

Minimotifs are short peptide sequences within one protein, which are recognized by other proteins or molecules. While there are now several minimotif databases, they are incomplete. There are reports of many minimotifs in the primary literature, which have yet to be annotated, while entirely novel minimotifs continue to be published on a weekly basis. Our recently proposed function and sequence syntax for minimotifs enables us to build a general tool that will facilitate structured annotation and management of minimotif data from the biomedical literature.

RESULTS:

We have built the MimoSA application for minimotif annotation. The application supports management of …

Overexpression And Partial Purification Of Recombinant Human Serine Hydroxymethyltransferase: A Potential Anticancer Target, Suprit Singh Deol

Chemistry and Biochemistry

Serine Hydroxymethyltransferase (SHMT) is a member of the thymidylate synthase cycle that provides pyrimidine nucleotides to actively dividing cells, including cancer cells. The other two enzymes involved in this cycle have been targets for clinical anticancer drugs, suggesting that inhibitors of SHMT may also be used as clinical anticancer drugs. In order to test potential inhibitors against SHMT, human SHMT has been overexpressed in E. coli and partially purified by selective ammonium sulfate fractionation followed by anion-exchange fast protein liquid chromatography. A coupled assay using L-allo-threonine and alcohol dehydrogenase was used to identify active fractions, which were analyzed …

Terminal Deoxynucleotidyl Transferase: The Story Of A Misguided Dna Polymerase, Edward A. Motea, Anthony J. Berdis

Chemistry Faculty Publications

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal …

Non-Natural Nucleotides As Probes For The Mechanism And Fidelity Of Dna Polymerases, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some …

Conformational Motion Associated With Catalysis In Indole-3-Glycerol Phosphate Synthase From S. Solfataricus, Tomasz Kurcon

Theses, Dissertations and Culminating Projects

Indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (SsIGPS) belongs to a broad family of (βα)₈-barrel enzymes. It catalyzes the fifth step in tryptophan biosynthesis, converting l-(o-carboxylphenylamino)-l-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). Site selective mutagenesis was used to introduce a single cysteine in two loops near the active site, generating two recombinant proteins, each containing a single cysteine handle. The two construct were labeled, each with two different thiol-reactive probes generating four labeled constructs that were used for this study. Steady-state kinetic parameters of the labeled mutants and the wild type SsIGPS were well characterized using fluorescence spectroscopy. Subsequent experiments …

A Novel Filarial Parasite (B. Malayi) Stress-Activated Protein Kinase As A Potential Drug Target, Akruti Patel

Theses, Dissertations and Culminating Projects

Lymphatic filariasis (or elephantiasis) is a major neglected disease with an estimated 120 million individuals infected and approximately 1.5 billion at risk in endemic regions. It is a highly disfiguring and debilitating disease and one of the major causes of global morbidity. Treatment options for this disease are few and new drug targets and therapies need to be identified. We have identified a protein kinase ortholog of human p38 mitogen-activated protein kinase (p38) expressed in the filarial parasite, Brugia malayi (B. malayi), one of three causative agent of lymphatic filariasis. We hypothesize that this protein kinase, BmMPKl, is important for …

Investigation Of Near-Infrared Fluorescence And Photobleaching Of Human Volar Side Fingertips In Vivo: Antioxidants And Melanin, Colin Wright

Renée Crown University Honors Thesis Projects - All

Noninvasive in vivo blood glucose determination in the skin of volar side of human fingertips by near-infrared (NIR) Raman spectroscopy relies on fluorescence to quantify blood volume. Fluorescence does not only come from blood, which is composed of plasma and red blood cells; in fact, most fluorescence produced by human fingertips originates in the static tissues, e.g. skin, interstitial fluid, etc.. It will soon be possible to quantify the precise contributions of red blood cells, plasma, and static tissue to the overall fluorescence emission. Observations reveal a systematic decay in fluorescence, which, if not caused by blood movement, challenges our …

The Role Of Cuticular Hydrocarbons In The Pre-Mating Isolation Of Two Pissodes Species, Stephanie L. Teale

Renée Crown University Honors Thesis Projects - All

Pissodes strobi and P. nemorensis are weevils (Coleoptera, Curculionidae) that infest pines (Pinus spp.) and spruces (Picea spp.). Previous studies indicate that they are able to hybridize. In the spring, breeding site specificity maintains reproductive isolation, but in the late summer, both species occupy lateral branches of host trees. Aggregation pheromones for P. nemorensis have been identified but the mechanism for late summer isolation is unknown. Cuticular hydrocarbons have been shown to play a role in the chemical recognition of species, sex, kin, and caste in many groups of insects. The large number of possible compounds and the …

Mtor In Cell Signaling And Size Enlargement As A Target For The Chemico-Physical Therapy Of Cancer, Gabriela Krawiec

Renée Crown University Honors Thesis Projects - All

mTOR is a kinase protein meaning it phosphorylates target proteins affecting their cell signaling properties¹. The drug Rapamycin, analogs of Rapamycin, and cell signaling proteins that interact with mTOR control the activities mediated by mTOR¹. mTOR is located in the cytoplasm at a convergent point of many signaling pathways that regulate a multiplicity of cellular processes including metabolism that precede cell enlargement (cell “growth”), cell proliferation (cell division), and angiogenesis^1,2. Cells with mTOR inappropriately activated can proceed with cell enlargement and cell proliferation in the absence of normal cell signaling². Rapamycin and …

The Effect Of Exogenous Estrogens On Primordial Follicle Assembly In Vivo, Jenna Rose Karavan

Renée Crown University Honors Thesis Projects - All

During embryogenesis in the mouse, primordial germ cells develop, move to the genital ridge of the embryo, and form germline cysts as the ovary develops. The cells in these cysts are linked by intercellular bridges. The cysts then undergo a breakdown process which ultimately results in primordial follicles, each of which consists of a single oocyte surrounded by somatic cells called granulosa cells. During this cyst breakdown process, approximately one-third of the original oocytes become enclosed in primordial follicles, the amount of which is representative of the number of eggs a female will have available to her during her reproductive …

Dna Polymerases: Perfect Enzymes For An Imperfect World, Anthony J. Berdis

Chemistry Faculty Publications

This Special Thematic Issue explores the molecular properties of DNA polymerases as extraordinary biological catalysts. In this short introductory chapter, I briefly highlight some of the most important concepts from the articles contained within this Special Issue. The contents of this Special Issue are arranged into distinct sub-categories corresponding to mechanistic studies of faithful DNA polymerization, studies of "specialized" DNA polymerases that function on damaged DNA, and DNA polymerases that are of therapeutic importance against various diseases. Emphasis is placed on understanding the dynamic cellular roles and biochemical functions of DNA polymerases, and how their structure and mechanism impact their …

Role Of Secondary Interactions (Π, Agostic) As Well As Solvent Influence On Alkali Metal Complexes, Elizabeth D. Nagle

Renée Crown University Honors Thesis Projects - All

We here attempt to synthesize heterobimetallic alkali metal complexes with bulky phenolate ligands via solid state direct metallation. The solid state route may offer a facile method of producing these heterobimetallic species. Synthesis of heterobimetallic alkali metal compounds remains a challenge, as the homometallic complexes are generally favored over formation of the heterobimetallic species. Previous work has shown that the alkali metals have a strong solvent dependency; the recrystallization of heterobimetallic products from toluene yields the heterobimetallic, while recrystallization from THF yields the homometallic. However, the ability for phenolate ligands to stabilize metal centers through secondary interactions, such as M∙∙∙π …

Understanding The Genetic And Molecular Mechanisms Of The Shl3 Phenotype In Arabidopsis Thaliana, Sukeerti Kesar

Renée Crown University Honors Thesis Projects - All

The study of plant genetics and the molecular mechanisms regulating plant-pathogen interactions is an intensely studied area of research in molecular genetics. Gaining an understanding of the defense mechanisms of plants has proven highly useful in the construction of transgenic plants for increased crop yield. The focus of my research was on the defense mechanisms of Arabidopsis thaliana, a plant in the mustard family. Several features of Arabidopsis make it an excellent model plant for molecular genetic studies. A few of these include a short life cycle (approximately 8 weeks), a completely sequenced genome, high fecundity, and the availability …

The Role Of A2b Adenosine Receptor Signaling In Adenosine Dependent Lung Disease, Yang Zhou

Dissertations and Theses (Open Access)

Chronic lung diseases and acute lung injuries are two distinctive pulmonary disorders that result in significant morbidity and mortality. Adenosine is a signaling nucleoside generated in response to injury and can serve both protective and destructive functions in tissues and cells through interaction with four G-protein coupled adenosine receptors: A1R, A2AR, A2BR, and A3R. However, the relationship between these factors is poorly understood. Recent findings suggest the A2BR has been implicated in the regulation of both chronic lung disease and acute lung injury. The work presented in this dissertation utilized the adenosine deaminase-deficient mouse model and the bleomycin-induced pulmonary injury …

Glycopeptidome Of A Heavily N-Glycosylated Cell Surface Glycoprotein Of Dictyostelium Implicated In Cell Adhesion, Christa L. Feasley, Jennifer M. Johnson, Christopher M. West, Catherine P. Chia

School of Biological Sciences: Faculty Publications

Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man8GlcNAc4 species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core R3-fucose. Site mapping confirmed the occupancy …

The Development Of A Comprehensive Mechanism For Intracellular Calcium Oscillations: A Theoretical Approach And An Experimental Validation, Amanda A. Borges, Deanna Salter, Sandor Kadar, Steven B. Symington

Pell Scholars and Senior Theses

Calcium is an important second messenger for cellular communication. Theoretical models help scientists understand its signalling mechanism. A comprehensive model was developed in order to minimize any limitations in the models currently presented in the literature. Experimental results support the model and therefore the theoretical model provides a plausible explanation of the dynamics of the calcium-signaling mechanism. In the future, additional verification will be performed using various experimental configurations on PC12 cells. Further, the model will be used to predict the response of cells to environmental factors such as pesticides and heavy metals.

Preeclampsia, Autoimmunity And The At1 Receptor, Roxanna A. Irani

Dissertations and Theses (Open Access)

Preeclampsia (PE) is a disease of late pregnancy characterized by maternal hypertension and proteinuria. It is associated with preterm delivery and significant perinatal morbidity and mortality. Despite affecting ~7% of first pregnancies, there is no effective screening method to identify women at risk, nor is there a definitive treatment other than delivery of the baby and placenta. Though the pathogenesis of PE remains unclear, an imbalance in the renin-angiotensin and immune systems are thought to be major contributors. Bridging these two concepts, it has recently been shown that women with PE harbor specific autoantibodies: the angiotensin II type 1 receptor …

Host-Pathogen Interactions Of Secreted And Surface Staphylococcus Aureus Factors, Vanessa Vazquez

Dissertations and Theses (Open Access)

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, …

The Ubiquitin Ligase Ube4b Is Required For Efficient Epidermal Growth Factor Receptor Degradation, Natalie Sirisaengtaksin

Dissertations and Theses (Open Access)

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane …

Investigation Of Biochemistry And Enzymology Of Acyl-Coenzyme A Synthetase, Yu Meng

All Dissertations

Acetyl-CoA, an essential metabolite at the junction of various anabolic and catabolic pathways, is generated both from the breakdown of carbohydrates, lipids, and amino acids, and from the activation of acetate. AMP-forming acetyl-CoA synthetase, a key enzyme for acetate activation into acetyl-CoA in all domains of life, is a member of the adenylate-forming enzyme superfamily. Although members in the superfamily have different biological functions, they all share the property of forming an acyl-adenylate intermediate. Here I describe my research on characterization of a medium-chain acyl-CoA synthetase from Methanosarcina acetivorans (Macs_Ma) and identification of key residues in acyl substrate …

Development Of Novel Methods And Their Utilization In The Analysis Of The Effect Of The N-Terminus Of Human Protein Arginine Methyltransferase 1 Variant 1 On Enzymatic Activity, Protein-Protein Interactions, And Substrate Specificity, Brenda Bienka Suh-Lailam

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first …

Antimicrobial Activity Of D-Lenolate®, Andy Phui

UNLV Theses, Dissertations, Professional Papers, and Capstones

Olive trees are one of the most important fruit trees in the Mediterranean. Although not validated by research, olive leaves are traditionally believed to fight off fever and infections. It has been shown that olive leaf extracts possess antimicrobial activity. Olive leaf extracts contain polyphenols. One of the major phenolic compounds is oleuropein. Oleuropein and other polyphenols have been shown to exhibit antimicrobial activity. East Park Research (EPR) developed an extraction process that they claim does not alter the chemical composition of the olive leaves. The extract is known by the commercial name d-lenolate®. Studies have provided evidence that d-lenolate®, …

Bacillus Cereus And Bacillus Anthracis Germination Kinetics: A Michaelis-Menten Approach, Helen Luu

UNLV Theses, Dissertations, Professional Papers, and Capstones

Bacillus species are rod-shaped, gram-positive bacteria that are capable of producing endospores. In this dormant stage, the endospores can persist in hostile physical and chemical environments. Once conditions become favorable, the spores germinate into actively dividing cells, vegetative cells. Germination is a crucial step for the pathogenicity of the Bacilli in affecting a host organism.

Our study applies mathematical approaches to spore germination to determine whether the binding of one germinant will affect the binding of another germinant. We pursued this approach with two different species, B. cereus and B. anthracis, both pathogenic organisms. B. cereus is a widely known …

Transesterification And Recovery Of Intracellular Lipids Using A Single Step Reactive Extraction, Daniel R. Nelson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A single-step, extractive reaction for extraction of lipids such as biodiesel components, omega-3 fatty acids, or other triglycerides from microbial cells was examined. Conventional methods for lipid extraction use toxic solvents, and require multiple steps and long processing times. When the goal is to produce fatty acid methyl esters or FAMEs, the extracted lipids are subjected to a separate transesterification reaction with simple alcohols in the presence of an acid or base catalyst. A simplified, single-step reactive extraction method can be applied that combines the sequential extraction followed by transesterification using acidified alcohols - a process known as in situ …

The Rho Family Gtpase: Determining Gef Specificity Through Recombinant Expression, Farrah Steinke

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Protein Evolution Via Amino Acid And Codon Elimination, Lise Goltermann, Marie Sofie Yoo Larsen, Ranat Banerjee, Andreas C. Joerger, Michael Ibba, Thomas Bentin

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background
Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.

Methodology/Principal Findings
The strategy involves combining a sequential …

Oxidation Of Methane By A Biological Dicopper Centre, Ramakrishnan Balasubramanian, Stephen M. Smith, Swati Rawat, Liliya A. Yatsunyk, Timothy L. Stemmler, Amy C. Rosenzweig

Biochemistry and Molecular Biology Faculty Publications

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol1. Current processes to activate the strong C–H bond (104 kcal mol−1) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst3. There are two types of MMO. Soluble MMO (sMMO),expressed by several strains of …

How The Sequence Of A Gene Can Tune Its Translation, Kurt Fredrick, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Sixty-one codons specify 20 amino acids, offering cells many options for encoding a polypeptide sequence. Two new studies (Cannarrozzi et al., 2010, Tuller et al., 2010) now foster the idea that patterns of codon usage can control ribosome speed, fine-tuning translation to increase the efficiency of protein synthesis.

Ribosomal Rna Mutations That Inhibit The Activity Of Transfer-Messenger Rna Of Stalled Ribosomes, Jacob N. Crandall

Theses and Dissertations

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required …

Dynamics On Multiple Timescales In The Rna-Directed Rna Polymerase From The Cystovirus Φ6, Zhen Ren, Hsin Wang, Ranajeet Ghose

Publications and Research

The de novoinitiating RNA-directed RNA polymerase (RdRP), P2, forms the central machinery in the infection cycle of the bacteriophage ϕ6 by performing the dual tasks of replication and transcription of the double-stranded RNA genome in the host cell. By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the δ1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps–ns) and slow (µs–ms) timescales. The characteristics of several motional modes including those that coincide with the catalytic timescale (500–800/s) are altered in the presence of …