Molecular Biology Commons^™

Extracellular Secretion Of A Cutinase With Polyester-Degrading Potential By E. Coli Using A Novel Signal Peptide From Amycolatopsis Mediterranei, Yeqi Tan, Gemma K. Kinsella, Gary T. Henehan, Barry J. Ryan

Articles

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis.

The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway.

E. …

Paddling Along The Voltage Gated Sodium Channel Galaxy With Sea Anemone Toxins: Structural Studies Of The Interaction Between The Paddle Motif From Nav1.5div And Sea Anemone Toxin, Adel K. Hussein

Dissertations, Theses, and Capstone Projects

Voltage gated sodium channels (VGSC) are membrane proteins that serve an important function in the central nervous system (CNS), peripheral nervous system (PNS), and cardiac muscles amongst others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Nine mammalian VGSC subtypes have been characterized, Na_V1.1 – Na_V1.9, that are expressed in a tissue specific manner, each with unique gating properties. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, long QT syndrome, and Brugada disease. Furthermore, these channels are one of the primary targets of …

Optimization And Standardization Of Dms Chemical Mapping Experiments For Probing Rna 3d Structure, Sarah Brady

Honors Theses

RNA plays a significant and crucial role in biological processes. The properties of RNA are ultimately responsible for each molecule’s unique function. The configuration and structural integrity of RNA is important for its functionality. Chemical mapping detailed in this thesis can be used to analyze the 3D structure of an RNA sequence. RNA is modified via methylation of adenine and cytosine bases using dimethyl sulfate, then reverse transcribed to DNA, then sequenced. The sites where the bases were methylated become mutations in the DNA sequence, allowing for identification of these locations when analyzing sequencing data. Conditions for modification can be …

Mimicking The Namib Desert Beetle With Hydrophilic And Hydrophobic Surfaces, Tyler Bracewell

Williams Honors College, Honors Research Projects

The Namib Desert beetle, Stenocara gracilipes, condenses fog that rolls in off the west coast of Namibia to provide itself with the water it needs for the day. By lifting its abdomen into the air to collect the water vapor, it condenses on hydrophilic bumps that are about 500μm in diameter and are spaced apart about 500-1500μm. Once the water reaches a critical size, the droplet rolls off the hydrophilic bump and onto the flat hydrophobic surface of the elytra. Since the beetle is angled with its abdomen in the air, the water rolls down to its mouth. A …

Elucidating The Proteasomal Regulatory Mechanism Of Proteasome Activator Pa28Γ /Regγ, Taylor Ann Thomas

Graduate Theses, Dissertations, and Problem Reports

Virtually all cellular processes are precisely regulated by the proteasome which is the primary enzyme responsible for the degradation of misfolded, damaged, or no longer necessary soluble proteins. To prevent any untimely degradation of these target protein substrates and protect the cell, the proteasome is tightly regulated via adaptor proteins, known as proteasomal regulators. There are many classes of proteasomal regulators each with their own unique structures, functions, and effects on protein degradation through the proteasome. One such class is the 11S family of proteasomal regulators which are also referred to as PA26/28, or REG. The 11S family are ATP-independent …

Mhc Diversity Analysis Of Spheniscus Demersus For In Situ And Ex Situ Populations, Antonieta Van Den Berg Monsalve

Undergraduate Honors Thesis Projects

The South African Penguin has been listed as endangered by the International Union for Conservation of Nature. Major histocompatibility complex (MHC) alleles provide valuable statistics because of their variation in terms of vertebrate genomes and are pertinent to immune and reproductive health. A greater number of MHC alleles correlates with survivability of a population because the genes control the immune and reproductive systems. Legacy research by Otterbein alumni and Dr. Simon Lawrance studied MHC samples from wild, in situ, and captive, ex situ, penguin populations. By comparing these populations' major histocompatibility complexes through biostatistical analysis, contributions to conservation of the …

The Role Of Charge On Dna Packaging And Integrity Within Reconstituted Peptide-Dna Assemblies, Ehigbai Oikeh

Theses and Dissertations--Chemistry

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20^th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, …

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …

Cryo-Em Structure Of Mechanosensitive Channel Ynai Using Sma2000: Challenges And Opportunities, Claudio Catalano, Danya Ben-Hail, Weihua Qiu, Paul Blount, Amedee Des Georges, Youzhong Guo

Publications and Research

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. Highresolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, …

A Nosy Neighbor: Purification And Functional Characterization Of Lpg2149, Ashley M. Holahan

The Journal of Purdue Undergraduate Research

Ubiquitination is a process that marks proteins for various cell-signaling pathways, namely protein degradation and other processes. Th ese pathways are essential in a wide array of cellular processes, including defense mechanisms against invading pathogens. Th e ubiquitination process is universally found in all eukaryotic organisms, including plants and animals, and thus plays a vital role in cellular homeostasis. Recently, more discoveries have been made on prokaryotic effector proteins that hijack the ubiquitination system even when they do not possess a ubiquitin system of their own. MavC, also known as lpg2147 (Gan, Nakayasu, Hollenbeck, & Luo, 2019; Puvar et al., …

Utilizing Fluorescence Microscopy To Characterize The Subcellular Distribution Of The Novel Protein Acheron, Varun Sheel

Masters Theses

All cells carry the genetic machinery required to commit cell suicide; a process known as programmed cell death (PCD). While the ability to initiate PCD serves a number of useful purposes during development and homeostasis, misregulation of PCD is the underlying basis of most human diseases, including cancer, autoimmunity disorders and neurodegeneration. Using the tobacco hawkmoth Manduca sexta as a model organism, the Schwartz lab at UMass has demonstrated that PCD requires de novo gene expression and has cloned many death-associated genes. One of these genes encodes a novel protein that was named Acheron after one of the rivers of …

An Empirical Pipeline For Personalized Diagnosis Of Lafora Disease Mutations, M. Kathryn Brewer, Maria Machio-Castello, Rosa Viana, Jeremiah L. Wayne, Andrea Kuchtová, Zoe R. Simmons, Sarah Sternbach, Sheng Li, Maria Adelaida García-Gimeno, Jose M. Serratosa, Pascual Sanz, Craig W. Vander Kooi, Matthew S. Gentry

Molecular and Cellular Biochemistry Faculty Publications

Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical …

Covid-19 In Silico Drug With Zingiber Officinale Natural Product Compound Library Targeting The Mpro Protein, Renadya Maulani Wijaya, Muhammad Aldino Hafidzhah, Viol Dhea Kharisma, Arif Nur Muhammad Ansori, Arli Aditya Parikesit

Makara Journal of Science

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic. Ginger (Zingiber officinale) is a rhizome, which is commonly used for culinary and medicinal purposes. In Indonesia, ginger is taken as traditional medicine by processing it into a drink known as jamu. The present study aimed to assess and evaluate the bioactive compounds in ginger that can be used in drug design for treating COVID-19. The crystal structure of the SARS-CoV-2 main protease (M^pro) was generated from a protein sequence database, i.e., Protein Data Bank, …

Dual Control Of One Component Signaling: Mechanistic And Structural Insights Into El222 Active States, Uthama Phani R. Edupuganti

Dissertations, Theses, and Capstone Projects

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the …

The Structural And Functional Role Of Photosensing In Rgs-Lov Proteins, Zaynab Jaber

Dissertations, Theses, and Capstone Projects

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion …

Modulation Of Protein Dynamics By Ligand Binding And Solvent Composition, Richard J. Lindsay

Doctoral Dissertations

Many proteins undergo conformational switching in order to perform their cellular functions. A multitude of factors may shift the energy landscape and alter protein dynamics with varying effects on the conformations they explore. We apply atomistic molecular dynamics simulations to a variety of biomolecular systems in order to investigate how factors such as pressure, the chemical environment, and ligand binding at distant binding pockets affect the structure and dynamics of these protein systems. Further, we examine how such changes should be characterized. We first investigate how pressure and solvent modulate ligand access to the active site of a bacterial lipase …

Awegnn: Auto-Parametrized Weighted Element-Specific Graph Neural Networks For Molecules., Timothy Szocinski, Duc Duy Nguyen, Guo-Wei Wei

Mathematics Faculty Publications

While automated feature extraction has had tremendous success in many deep learning algorithms for image analysis and natural language processing, it does not work well for data involving complex internal structures, such as molecules. Data representations via advanced mathematics, including algebraic topology, differential geometry, and graph theory, have demonstrated superiority in a variety of biomolecular applications, however, their performance is often dependent on manual parametrization. This work introduces the auto-parametrized weighted element-specific graph neural network, dubbed AweGNN, to overcome the obstacle of this tedious parametrization process while also being a suitable technique for automated feature extraction on these internally complex …

Molecular Dynamics Simulations Provide Insight Into Stability Of Hyperthermophilic Endoglucanases, Logan E. Sheffield

Electronic Theses and Dissertations

Endoglucanases play a key role in the industrial production of bioethanol, but the most efficient method requires the utilization of high temperatures and is currently limited by the thermostability of endoglucanases. For this reason, it would be beneficial to discover more high-efficiency, thermostable enzymes to utilize in the hydrolytic process. In this study molecular dynamics simulations were performed on structurally similar endoglucanases with varying levels of thermostability to gain insight on what factors contribute to thermostability in endoglucanases. RMSD, RMSF, PCA, hydrogen bonding and salt bridges were analyzed. Finally, protein energy networks were constructed from nonbonded interaction potentials and analysis …

Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu

Doctoral Dissertations

Human lysosomal neuraminidase 1 (hNEU1) is an exo-a-sialidase which cleaves a(2-3) and a(2-6) linked sialic acids on glycoproteins in the lysosome. Deficiency of hNEU1 in the lysosome results in sialidosis, a lysosomal storage disease. Currently there is no effective treatment for sialidosis, which leads to a rising interest in discovering potential therapies. Here we presented a small molecule, α-D-N-acetylneuraminic acid (NANA), increases the protein amount and activity of both wild-type hNEU1 and three different hNEU1 mutations found in sialidosis patients in our mammalian cell system, suggesting that NANA works as a potential pharmacological chaperone for hNEU1 and provides …

Search For Palladin, An Actin-Associated Protein, In Pig Retinal Pigmented Epithelium And Its Role In Epithelial-Mesenchymal Transition, Katrina Powell

Undergraduate Theses

This study investigates the expression of Palladin, a phosphoprotein product of the PALLD gene, in the retinal pigmented epithelium (RPE). Palladin is an actin cross-linking protein and plays a role in cell adhesion and motility. Published reports have demonstrated that a down regulation of Palladin in colon cancer cells results in a reorganization of the actin cytoskeleton, causing the cells to lose their typical shape, become proliferative and migratory. This process is otherwise known as epithelial-mesenchymal transition (EMT). A similar EMT phenomenon is observed when the RPE is exposed to the vitreous humor in patients with proliferative vitreoretinopathy (PVR). In …

Mitochondrial Distribution Of Glycine Receptors In Motor Neuron Cell Lines, Katsiaryna Milashevich

Student Theses and Dissertations

Although non-essential, glycine plays an important role in major metabolic reactions and is most known for its anti-inflammatory effects. An accumulation of contemporary research has shown that glycine is able to stabilize membrane potential using glycine receptors at the cellular level and to protect mitochondrial function directly, whether it is from inflammation, heavy metal poisoning, or ischemia-induced neuroinflammation. In this research, the existence of a hypothetical mitochondrial glycine receptor is examined. Immunofluorescence imaging was used to examine the presence of the glycine receptor subunits alpha 1 and alpha 2 in both non- differentiated and differentiated neuroblastoma cell lines. The preliminary …

Lighting The Way: Recent Insights Into The Structure And Regulation Of Phototropin Blue Light Receptors, Jaynee E. Hart, Kevin H. Gardner

Publications and Research

The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two bluelight- sensing Light–Oxygen–Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made …

Insights Into Viral Genome Function Through Comparative Structural Analysis, Lydia Phillips

UNO Student Research and Creative Activity Fair

Enteroviruses are single stranded RNA viruses which have caused many public health concerns, particularly in children. These viruses are responsible for polio, hand, foot, and mouth disease, many polio-like neurological diseases, and the common cold. The enterovirus called Coxsackievirus B3 (CVB3), a close relative to poliovirus, has been shown responsible for severe human disease, including pancreatitis, myocarditis, and type 1 diabetes. A unique advantage of studying CVB3 is the existence of a naturally occurring strain (CVB3/GA) which displays no known pathogenicity. Earlier comparative genomic studies have shown that the primary difference between avirulent CVB3/GA and the virulent strains of CVB3 …

'Induction Of Apoptosis, Cytotoxicity And Radiosensitization By Novel 3,4-Dihydroquinazolinone Derivatives, Eman Ramadan, Amira Khalil

Pharmacy

Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14 and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target compounds showed IC₅₀ in the range of 2.46-36.85 µM and 3.87-88.93 µM for HepG-2 and MCF-7, respectively. The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC₅₀ values of the promising compounds were in the range of 146.9-1032.7 nM for EGFR in …

Photoreceptor Phosphodiesterase (Pde6): Structure, Regulatory Mechanisms, And Implications For Treatment Of Retinal Diseases, Rick H. Cote, Richa Gupta, Michael J. Irwin, Xin Wang

Faculty Publications

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signaling-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes in a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Photoreceptor Phosphodiesterase (Pde6): Activation And Inactivation Mechanisms During Visual Transduction In Rods And Cones, Rick H. Cote

Faculty Publications

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary

intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes …

On The Structure And Function Of Mitochondrial Uncoupling Proteins: The Case Of Ucp2, Afshan Ardalan

Theses and Dissertations (Comprehensive)

Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins …

The Shape Of U: Mapping Out Protective Elements In Mrna Escapees, Jacob Miles

Masters Theses

A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the …

Rna Structure And Function: Biological Relevance In Neurodegenerative And Infectious Disease Pathogenesis, Joshua Imperatore

Electronic Theses and Dissertations

The studies outlined in this dissertation encompass a broad focus, relating to the pathogenesis of neurological disorders such as Alzheimer’s disease, fragile X syndrome, and amyotrophic lateral sclerosis, as well as a novel infectious disease, COVID-19. However, all four studies detailed within this dissertation contain similar elements, namely, the coordinated role of RNA structure and function in essential molecular mechanisms. The results of these investigations allow us to speculate on various disease- related mechanisms, including dysregulated microRNA processing pathways, nucleocytoplasmic shuttling via distinct RNA-protein interactions, and fine-tuned molecular switches controlling genomic dimerization and viral hijacking of host cellular microRNAs. Taken …