Biochemistry Commons^™

P-20 Characterization Of Cgx Tautomerase And Mutants With Acetylenecarboxylic Acid, Daniel Chi, Karen Draths, Amaya Sirinimal

Celebration of Research and Creative Scholarship

In the production of commodity chemicals through microbial synthesis, the use of renewable sugars as a raw material is increasingly common. However, this generates competition between its use in the chemical industry and the food industry. Therefore, developing alternative biosynthetic pathways involving other renewable feedstocks to replace starch-derived feedstocks is crucial. CgX, a tautomerase native to Corynebacterium glutamicum, catalyzes the hydration and subsequent decarboxylation of acetylenecarboxylic acid (ACA), a compound that can be produced from methane gas and carbon dioxide, both abundant greenhouse gases. The products formed from the CgX (wild-type)-catalyzed reaction of ACA results in a mixture of …

The Essential Role Of Carbon Metabolism In The Virulence Of Cryptococcus Neoformans, Mara Weigner

Senior Honors Theses

Cryptococcus neoformans infections are a major cause of meningoencephalitis in immunosuppressed patients worldwide. Inhaled as spores or desiccated yeast cells, C. neoformans can undergo metabolic changes in response to the new host environment that allow it to cross the blood brain barrier and cause deadly central nervous system (CNS) infections. Nutrient acquisition, and specifically carbon metabolism, is critical for survival and proliferation within the host. Notably, efficient carbon metabolism is necessary to produce the polysaccharide capsule, which is arguably C. neoformans’ most important and well-studied virulence factor. As such, a better understanding of carbon acquisition and regulation is essential for …

Bright Green Biofluorescence In Sharks Derives From Bromo-Kynurenine Metabolism, Hyun Bong Park, Yick Chong Lam, Jean P. Gaffney, James C. Weaver, Sara Rose Krivoshik, Randy Hamchand, Vincent Pieribone, David F. Gruber, Jason M. Crawford

Publications and Research

Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as …

The Fitness Landscape Of The African Salmonella Typhimurium St313 Strain D23580 Reveals Unique Properties Of The Pbt1 Plasmid, Rocío Canals, Roy R. Chaudhuri, Rebecca E. Steiner, Siân V. Owen, Natalia Quinones-Olvera, Melita A. Gordon, Michael Baym, Michael Ibba, Jay C. D. Hinton

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These …

Creating A Molecular Map Of The Pediatric Lung, Quinlen F. Marshall

Forum Lectures

The newborn lung undergoes vast biochemical and physiological changes during adaptation from the intrauterine to the extrauterine environment. Lung morphogenesis continues from birth into early childhood, mediated by dynamic gene expression and a diversity of pulmonary cell types that exhibit remarkable heterogeneity. (Whitsett, JA. et al. Physiol. Rev, 2019). Surprisingly, few studies have solely focused on human lung development during this critical period, and many current studies of lung maturation rely on adult, murine, or diseased samples, limiting their insights and applicability to longitudinal pediatric lung development. Understanding the molecular and physiological nuances of pulmonary development has important clinical relevance, …

Developing A Dissociative Nanocontainer For Peptide Drug Delivery, Michael Patrick Kelly

Dissertations, Theses, and Capstone Projects

The potency and specificity of bioactive peptides have propelled these agents to the forefront of pharmacological research. However, delivery of peptides to their molecular target in cells is a major obstacle to their widespread application. A Trojan Horse strategy of packaging a bioactive peptide within a modified protein cage to protect it during transport, and releasing it at the target site, is a promising delivery method. Recent work has demonstrated that the viral capsid of the P22 bacteriophage can be loaded with an arbitrary, genetically-encoded peptide, and externally decorated with a cell-penetrating peptide, such as HIV-Tat, to translocate across in …

Innate Antibodies, Murine Models, And Evolution: A Study Of Trypanosome Lytic Factor Functions And Their Translational Applications, Joseph P. Verdi

Dissertations, Theses, and Capstone Projects

Trypanosome lytic factors (TLFs) are primate-specific antimicrobial protein complexes that lyse African trypanosome parasites by delivering the channel-forming toxin APOL1 to the invading microorganisms. Human serum contains two TLFs that are delivered to the parasite by separate mechanisms, only one of which has been characterized. TLF1 is endocytosed by a receptor that is typically blocked by other serum factors in vivo, suggesting that TLF2 is the more relevant lytic factor in the context of trypanosome immunity. TLF2 is non-covalently associated with polyclonal immunoglobulin M (IgM) antibodies, which we report here to be involved in the uptake mechanism. The TLF2-IgMs …

Engineering Of Escherichia Coli 2-Oxoglutarate Dehydrogenase Complex With Mechanistic And Synthetic Goals, Joydeep Chakraborty

Dissertations

The Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) compromises multiple copies of three enzymes - 2-oxoglutarate dehydrogenase (E1o), dihydrolipoyl succinyltransferase (E2o), and dihydrolipoyl dehydrogenase (E3). OGDHc is found in the Krebs cycle and catalyzes the formation of the all-important succinyl-Coenzyme A (succinyl-CoA). OGDHc was engineered to understand the catalytic mechanism and optimized for chemical synthetic goals.

Succinyl-CoA formation takes place within the catalytic domain of E2o via a transesterification reaction. The succinyl group from the thiol ester of S8-succinyldihydrolipoyl-E2o is transferred to the thiol group of CoA. Mechanistic studies were designed to investigate enzymatic transthioesterification. His375 and Asp374 was shown to …

Characterization Of Endothelial Nitric Oxide Synthase Serine-600 Phosphorylation, Kevin Patel

Master of Science in Chemical Sciences Theses

Endothelial nitric oxide synthase (eNOS) is part of a family of three nitric oxide synthase (NOS) enzymes that catalyze the production of nitric oxide (NO). NO is a gaseous, free-radical signaling molecule that has a variety of cellular and physiological functions that range from maintaining cardiovascular homeostasis to neurotransmission. The function of NO greatly depends on the concentration and is cell type specific. eNOS is the most regulated of the three NOS isoforms and the mechanisms of regulation can be through protein-protein interactions and posttranslational modifications. A connection with eNOS and the cell cycle has begun to form with recent …

How To Untie A Protein Knot., Sitao Yin, Brittiny Dhital, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

The origin of protein backbone threading through a topological knot remains elusive. To understand the evolutionary origin of protein knots, in this issue of StructureKo et al. (2019) used circular permutation to untie a knotted protein. They showed that a domain-swapped dimer releases the knot and the associated high-energy state for substrate binding.

Iron-Sulfur Cluster Assembly; In Vivo Analysis Of The Methanogenic Suf System, Evan Dunkle

LSU Master's Theses

Iron-sulfur (Fe-S) clusters are among the most ancient and prevalent of all biological cofactors. Their assembly into associated proteins is a tightly regulated process with many organisms employing multiple cluster assembly pathways. Much is known about Fe-S cluster assembly in aerobic organisms such as Escherichia coli (E. coli) but little is known in regards to cluster assembly in more ancient organisms such as methanogens. Methanogens are members of the domain of Archaea and are defined by their ability to generate methane as a byproduct of their main energy generating pathway. Methanogens also have significantly higher Fe-S cluster content …

Temporal Gene Expression Of Mesenchymal Cells In The Pediatric Lung, Quinlen F. Marshall, Soumyaroop Bhattacharya, Gautam Bandyopadhyay, Ravi Misra, Thomas Mariani, Gloria Pryhuber

Chemistry Student Work

INTRODUCTION: The newborn lung undergoes vast biochemical and physiological changes during adaptation from the intrauterine to the extrauterine environment. Lung morphogenesis continues from birth into early childhood, mediated by dynamic gene expression and a diversity of pulmonary cell types (Whitsett, JA. et al. Physiol. Rev, 2019). Murine models demonstrate that pulmonary mesenchymal cells exhibit remarkable heterogeneity in function and morphology during development, however, confirmation of their role is lacking in human neonates and early childhood (Guo, M. et al. Nat. Comm, 2019). In addition, many current human genomic studies of lung maturation suffer from limited sample size, limiting …

The Dynamin-Like Protein Vps1 Stimulates Endosome-To-Golgi Fusion In Vitro, Jared Christopher Smothers

Graduate Theses/Dissertations

Intracellular membrane fusion events can be reconstituted by exploiting isolated organelles from cellular hosts or artificial membranes made of purified phospholipid components. Artificial construction of membranes provides two significant advantages. First, cellular isolation of the endosome-derived vesicles and TGN (trans-Golgi Network) compartments needed for the fusion assay would be extremely challenging. Second, reconstituting the membranes provides the added benefit of controlling size and lipid compositions to functionally mimic the individual membrane architectures and introduce only the purified proteins that are under investigation. For these reasons, I have developed the first simultaneous lipid and content mixing fusion assays that measures the …

Does Thermotolerance In Daphnia Depend On The Mitochondrial Function?, Rajib Hasan

Electronic Theses and Dissertations

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18^ᵒC and 25^ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP …

The Gsk-3Β-Fbxl21 Axis Regulates Tcap Via Ubiquitin-Mediated Proteasomal Pathway In The Cytoplasm, Jiah Yang

Dissertations and Theses (Open Access)

Protein turnover is one of the most essential mechanisms controlling circadian rhythms. F-Box and Leucine Rich Repeat Protein21 (FBXL21) is a circadian E3 ligase which shows oscillatory mRNA transcripts and protein levels. It was previously found to perform subcellular compartment-specific E3 ligase activities targeting the core clock proteins CRYPTOCHROME(CRY)1/2. Here we identified a new sarcomeric target substrate, Telethonin(TCAP), which also shows circadian oscillation in its mRNA transcript and protein expression and, importantly, interaction with FBXL21 in an anti-phasic manner. Via computational and pharmacological tests, we identified Glycogen Synthase Kinase-3β(GSK-3β) as a regulator of FBXL21. Biochemical and molecular characterizations demonstrated that …

The Distinct Expressions Of Integrins Αdβ2 And Αmβ2 Differently Regulate Macrophage Migration In 3d Matrix In Vitro And In Tissue During Inflammation, Kui Cui

Electronic Theses and Dissertations

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins α_Dβ₂ and α_Mβ₂ to macrophage migration and development of chronic inflammation.

We found that integrin α_Dβ₂ is upregulated on M1-macrophages in vitro and …

Obscurin Mediates Ankyrin Complex Formation In The Heart, Janani Subramaniam

Dissertations and Theses (Open Access)

Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins are crucial to cardiac function. Interactions between adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes. Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as arrhythmias and cardiomyopathies. Alternative splicing of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies …

Investigations Of The Structure-Function Relationship In Kainate Receptors Using FöRster Resonance Energy Transfer, Douglas Litwin

Dissertations and Theses (Open Access)

Kainate receptors belong to the family of ion channels known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission, modulate the release of presynaptic glutamate, and facilitate dendrite formation. Kainate receptors are unique among the ionotropic glutamate receptors in being modulated by sodium ions. They have also been implicated in the development of higher learning and epilepsy. In recent years a wealth of structural data has become available for the AMPA and NMDA classes; however, the structural characterization of kainate receptors has been limited. The work in this dissertation utilizes luminescence resonance energy transfer …

Translational Control Of Antibiotic Resistance, Anne Witzky, Rodney Tollerson Ii, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Many antibiotics available in the clinic today directly inhibit bacterial translation. Despite the past success of such drugs, their efficacy is diminishing with the spread of antibiotic resistance. Through the use of ribosomal modifications, ribosomal protection proteins, translation elongation factors and mistranslation, many pathogens are able to establish resistance to common therapeutics. However, current efforts in drug discovery are focused on overcoming these obstacles through the modification or discovery of new treatment options. Here, we provide an overview for common mechanisms of resistance to translation-targeting drugs and summarize several important breakthroughs in recent drug development.

Iron-Sulfur Cluster Biosynthesis In Methanogens, Cuiping Zhao

LSU Doctoral Dissertations

Methanogens live in a syntrophic consortium with bacteria, taking advantage of the metabolic abilities of their syntrophic partners to overcome energetic barriers and break down compounds that they cannot digest by themselves. Interspecies electron transfer, which is a major type of microbial communication in syntrophic processes, improves methanogenesis and anaerobic oxidization of methane (AOM) processes involved in syntrophic consortia. These processes have a significant impact on the global carbon cycle. Most of the essential enzymes involved in methanogenesis are iron-sulfur proteins. Iron-sulfur clusters are one of the oldest and most versatile cofactors present in all domains of life. To date, …

Divergent Transcriptional Regulation Of Suppressors Of Cytokine Signaling Genes In Adipocytes, Paula Mota De Sa

LSU Doctoral Dissertations

The Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway transduces several signals crucial for development and homeostasis. Suppressors of cytokine signaling (SOCS) proteins control JAK-STAT signaling via a negative feedback loop. The transcription factor STAT5 is known to play a significant role in fat cell development and function, and several studies suggest that acetylation may affect STAT5 transcriptional activity. To test this hypothesis, we treated 3T3-L1 adipocytes with growth hormone (GH) to activate STAT5 in the presence or absence of histone deacetylase (HDAC) inhibitors. STAT5 acetylation levels were low in adipocytes and mostly unchanged by the …

In Vitro Evaluation Of Ovarian Cancer Tumorigenesis As A Function Of Quinone Oxidoreducatse-1 And Cell Phenotype, Milcah S. Jackson

LSU Doctoral Dissertations

In vitro multicellular spheroids are attractive model systems for assessing genetic and epigenetic changes that occur in diseased tissues. Understanding how such alterations in gene and subsequent protein expression affect disease progression and metastasis, drug resistance, and recurrence is of great interest in cancer research. In this regard, examining expression and activity of proteins, such as those with cytoprotective ability that are overexpressed in cancer cells, in addition to cell phenotype (i.e., stem-like, epithelial, mesenchymal, or mixed), are two ways to evaluate genetic and epigenetic changes. Moreover, determining the impact that cytoprotective proteins and cell phenotype have on tumor formation …

Application Of Single Particle Electron Microscopy To Native Lens Gap Junctions And Intrinsically Disordered Signaling Complexes, Janette Bernadette Myers

Dissertations and Theses

Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part …

Arachidin 3 Modulation Of Lipid Metabolism In Rotavirus Infections, Stormey Wisdom

Electronic Theses and Dissertations

Rotavirus (RV) can cause severe and deadly gastroenteritis in young children, infants, and immunocompromised individuals. Previous studies have shown that arachidin 3 (A3) inhibits RV replication, and that RV replication is dependent on the presence of lipids. This study investigated the alteration of lipid metabolism by A3 in RV infected HT29.f8 cells. A decrease in the RV regulation of lipid biosynthesis genes was observed with the addition of A3 using qRT-PCR. Also, immunofluorescent and histochemical staining for neutral fats, a major component of cellular lipid droplets, revealed an increased accumulation with both RV and RV+A3 when compared to no virus …

Modulation Of Biological Responses To 2 Ns Electrical Stimuli By Field Reversal, Esin B. Sözer, P. Thomas Vernier

Bioelectrics Publications

Nanosecond bipolar pulse cancellation, a recently discovered Phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (> 100 mu s) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). …

Nf-Κb In Biomphalaria Glabrata: A Genetic Fluke?, Paige Stocker

Lawrence University Honors Projects

Biomphalaria glabrata is the intermediate host to the disease causing parasitic worm, Schistosoma mansoni. Previous work has identified homologs of NF-κB, a known immune related transcription factor, in B. glabrata and work has also been done to establish putative κB sites. It has also been observed that the p65 homologous subunit has an extended N-terminal region not present in other homologs. The goal of the present study is twofold: investigate DNA binding affinity of two NF-κB subunits, Bg-p65 and Bg-p50, and characterize the nature of the N-terminal extension of Bg-p65. In the current work, it is shown through the …

Understanding How Map Kinases Influence Endothelial Nitric-Oxide Synthase Activity, Xzaviar Solone

Master of Science in Integrative Biology Theses

Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at Ser¹¹⁴ contrasts with ERK, which phosphorylated Ser⁶⁰⁰, and p38, which phosphorylated …

Molecular Signatures Of Calpain 10 Isoforms Sequences, Envisage Functional Similarity And Therapeutic Potential, Bushra Chaudhry, Farina Hanif, Kausar Saboohi

Department of Biological & Biomedical Sciences

Calpain 10 plays a role in insulin secretion, action and susceptibility to type 2 diabetes. The mechanism through which it influences the insulin secretion and action is not completely defined. A structural bioinformatics approach is applied to envision its mechanism of action using available tools on NCBI (blastp and blastn), EMBL-EBI, Ensembl, Swiss Model Repository websites, I-TASSER, PROCHECK program and Discovery Studio software. Homology of domain I and II of calpain10 (isoform a) was established with super family cysteine proteinase domains (II a and II b, e=1.30e-77, 1.00e-20). Remaining sequences of domain III and T from (isoform a and c) …

Role Of The Drosophila Beaf Protein In Chromatin Domain Insulator And Promoter Function, Mukesh Maharjan

LSU Doctoral Dissertations

Proper folding of eukaryotic genomes is required to allow correct interactions between different parts of chromosomes. Precise and timely interactions among different parts of a chromosome allow proper functioning inside a nucleus, including gene regulation, DNA replication and DNA repair. Eukaryotic regulatory elements that facilitate folding and interactions include enhancers, promoters and insulator elements. Insulator elements and their binding proteins play an important role in regulating correct chromatin structure and function. The Drosophila melanogaster special chromatin structure (scs’) is one such insulator. The Boundary Element Associated Factor (BEAF) binds to scs’. BEAF is a 32 kDa protein that has two …

Identification Of Pathways Required For The Survival Of Inversion(16) Acute Myeloid Leukemia, Yiqian Wang

Theses & Dissertations

Inversion of chromosome 16 [inv(16)] acute myeloid leukemia (AML) generates a fusion gene CBFB-MYH11. Approximately half of inv(16) AML patients eventually relapse mainly due to the existence of leukemia stem cells (LSCs). Previous work using a Cbfb-MYH11 knockin mouse model showed that the LSCs are enriched within CSF2RB^- population. Another gene upregulated by Cbfb-MYH11 encodes the cytokine receptor IL1RL1. Using Cbfb-MYH11 knockin mice, we showed that LSCs exist in multiple sub-populations defined by their immunophenotype, and IL1RL1 is expressed by cell populations with high LSC activity. We also found that treatment of IL-33, the ligand for IL1RL1, promoted …