Biochemistry, Biophysics, and Structural Biology Commons^™

Effects Of The Loss Of Multidrug Resistance Associated Protein 1 On Steroid Homeostasis, Dendritic Cell Function And Compensatory Mechanisms, Jeffrey Charles Sivils

Open Access Theses & Dissertations

The ATP-binding cassette (ABC) superfamily of membrane transporters use energy derived from ATP to eliminate a variety of exogenous and endogenous compounds from cells including anti cancer and anti viral drugs, metals steroids, bilirubin, cAMP, cGMP, leukotrienes , prostaglandins. The multi drug resistance associated protein family (MRP/ABBC) has been acknowledged as a major player involved in multi drug resistance (MDR), in which cancers stop responding a wide array of structurally and functionally unrelated chemotherapy drugs. Cancer is the second leading cause of death in adults in the United States. Although there have been great strides made the treatment of cancers …

Interactions Of The Cellular Sumoylation System With Influenza A Virus And Its Non-Structural Protein Ns1a (Ns1a), Sangita Pal

Open Access Theses & Dissertations

The most important current anti-influenza weapons, vaccination and antiviral drugs, can be rapidly rendered fully ineffective thanks to the virus's high mutational rate, which produces viruses exhibiting new antigenic properties and structural proteins insensitive to the drug's mechanism of action. One attractive alternative is to develop drugs that modulate the activity of cellular systems either required for viral growth or able to neutralize viral growth. Here we demonstrate that the cellular SUMOylation system, a post-translational modification involving the conjugation of the Small Ubiquitin-like MOdifier (SUMO) to specific protein targets using a Ubiquitin-like enzymatic cascade, interacts closely with influenza virus during …

The Role Of Sumoylation Of Ledgf/P75 In Hiv-1 Infection, Murilo Tadeu Domingues Bueno

Open Access Theses & Dissertations

The lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional co-activators that protect cells from stresses through modulation of stress and heat shock-related genes. Besides regulating such genes, LEDGF/p75 is also important in the process of HOX gene expression and leukemia transformation driven by the MLL histone methyl transferase complex. By exploiting a similar mechanism of interaction between LEDGF/p75 and MLL, the HIV-1 viral protein Integrase (IN) associates with LEDGF/p75 in order to execute efficient viral DNA integration. This present work has identified that LEDGF proteins are posttranslationally modified by SUMO-1 and -3. SUMOylation was found to target …

Isolation, Characterization And Molecular Cloning Of Dnase Iiib From Drosophila Melanogaster, Brenda Cristina Anchondo

Open Access Theses & Dissertations

Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββα-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA non-specific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar …

Ganglioside-Cytokine Interaction In The Induction Of Primary Brain Cell Death, John Charles Gorbet

Open Access Theses & Dissertations

Gangliosides have been implicated in multiple pathologies affecting the central nervous system (CNS) and recent research has implicated them in playing an active role in the pathogenesis of multiple sclerosis. Empirical studies and theoretical considerations have suggested the possibility of interactions between gangliosides, like GD3, and pro-inflammatory cytokines present in the nervous system. This study sought to investigate the possibility that either individual gangliosides acting alone or complexed with other species interact with the known immune response factor TNF&alpha to initiate or facilitate cell death in the CNS. I examined the cellular viability and gene expression in primary brain cell …

Phosphorylation Of The Glycine Transporter 1, Javier Vargas Medrano

Open Access Theses & Dissertations

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the high-affinity glycine transporter 1 (GlyT1) and the clearance of glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the past years, it has been shown that PKC activation diminishes the activity and promoted phosphorylation of several neurotransmitter transporters including the dopamine, serotonin and norepinephrine transporters however, its role is unknown for the glycine transporter. To get insights into the role of PKC activation on GlyT1 regulation, we used three N-terminus GlyT1 isoforms stably expressed in porcine aortic endothelial (PAE) …

Evaluating The Role Of Evolutionarily Conserved Regions Of Ledgf/P75 In Hiv-1 Infection, Jose Adrian Garcia

Open Access Theses & Dissertations

LEDGF/p75 is an important cellular co-factor for lentiviral integration. LEDGF/p75-deficient cells are markedly resistant to HIV-1 infection and re-expression of the wild type protein rescues infectivity. Although the molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet known, this co-factor activity requires the interaction of LEDGF/p75 with both the host chromatin and viral integrase. In order to evaluate the involvement of other LEDGF/p75 regions in HIV-1 infection we constructed a panel of deletion mutants targeting clusters of charged residues that are evolutionarily conserved and predicted to be post-translationally modified. These mutants were evaluated for their ability to rescue HIV-1 …

Electric Pulses To Prepare Feeder Cells For Sustaining And Culturing Of Undifferentiated Embryonic Stem Cells, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Current challenges in embryonic-stem-cell (ESC) research include inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gammaradiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed-electric-fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10- nanosecond-electric-pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1s pulse interval, …

Influence Of Cultivation Conditions On The Production Of A Thermostable Extracellular Lipase From Amycolatopsis Mediterranei Dsm 43304, Dharmdendra Dheeman, Jesus Maria Frias, Gary Henehan

Articles

Among several lipase producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture condition and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28 °C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. In optimal culture conditions the lipase activity was enhanced 12-fold with a 2-fold increase in lipase specific activity. The …

Quantitative Nuclear Proteomics Identifies Mtor Regulation Of Dna Damage Response, Sricharan Bandhakavi, Young-Mi Kim, Seung-Hyun Ro, Hongwei Xie, Getiria Onsongo, Chang-Bong Jun, Do-Hyung Kim, Timothy J. Griffin

Department of Biochemistry: Faculty Publications

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. …

N-Glycans On The Link Domain Of Human Hare/Stabilin-2 Are Needed For Hyaluronan Binding To Purified Ecto-Domain, But Not For Cellular Endocytosis Of Hyaluronan, Ed Harris, Simon Parry, Mark Sutton-Smith, Madhu S. Pandey, Maria Panico, Howard R. Morris, Stuart M. Haslam, Anne Dell, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The hyaluronic acid receptor for endocytosis (HARE)/Stabilin- 2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ligand-binding sites are within the 190 kDa isoform, which contains ~25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N²²⁸⁰, within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, …

Functional Hybrid Rubisco Enzymes With Plant Small Subunits And Algal Large Subunits Engineered Rbcs Cdna For Expression In Chlamydomonas, Todor Genkov, Moritz Meyer, Howard Griffiths, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, …

Structure Of The Proline Utilization A Proline Dehydrogenase Domain Inactivated By N-Propargylglycine Provides Insight Into Conformational Changes Induced By Substrate Binding And Flavin Reduction, Dhiraj Srivastava, Weidong Zhu, William H. Johnson Jr., Christian P. Whitman, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it …

On The Mechanism Of Protein Fold-Switching By A Molecular Sensor, Margaret M. Stratton, S N. Loh

Biochemistry & Molecular Biology Department Faculty Publication Series

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Mechanistic Importance Of Redox Potentials And Conformational Flexibility In Electron-Transferring Flavoproteins, Michael Anthony Swanson

Electronic Theses and Dissertations

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated.

ETF-QO contains one [4Fe-4S]^2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring …

Synthesis And Evaluation Of Azetidinone Analogues Of Combretastatin A-4 As Tubulin Targeting Agents, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Orla Bergin, Seema M. Nathwani, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

Articles

The synthesis and antiproliferative activity of a new series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (β-lactam) ring system in place of the usual ethylene bridge present in the natural combretastatin stilbene products. These novel compounds are also substituted at position 3 of the β-lactam ring with an aryl ring. A number of analogues showed potent nanomolar activity in human MCF-7 and MDA-MB-231 breast cancer cell lines, displayed in vitro inhibition of tubulin polymerization and did not cause significant cytotoxicity in normal murine breast epithelial cells. 4-(4-Methoxyaryl)-substituted compound 32, 4-(3-hydroxy-4-methoxyaryl)-substituted compounds 35 and 41 and …

Uncultivated Environmental Tm7 Model To Study Human Disease-Associated Tm7 Bacteria, David Barton

Master's Theses

The TM7 bacterial phylum has no cultivated species and includes members that span a broad range of environmental and human habitats, some of which are associated with human periodontitis. In this project, activated wastewater TM7 bacteria were analyzed and their relatedness compared to human-associated TM7 bacteria for the long-term goal of using an environmental TM7 to better understand TM7 pathogenesis in humans. DNA was extracted from activated wastewater and PCR amplified using TM7 16S rRNA gene- specific primers. The ~1,170 base pair PCR products were then cloned and sequenced. DNA sequencing and phylogenetic analysis identified environmental TM7 clones with high …

15N Solid-State Nmr Detection Of Flavin Perturbation By H-Bonding In Models And Enzyme Active Sites, Dongtao Cui

University of Kentucky Doctoral Dissertations

Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the ¹⁵N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that …

In Vivo Oxidative Stress In Alzheimer Disease Brain And A Mouse Model Thereof: Effects Of Lipid Asymmetry And The Single Methionine Residue Of Amyloid-Β Peptide, Miranda Lu Bader Lange

University of Kentucky Doctoral Dissertations

Studies presented in this dissertation were conducted to gain more insight into the role of phospholipid asymmetry and amyloid-β (Aβ)-induced oxidative stress in brain of subjects with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). AD is a largely sporadic, age-associated neurodegenerative disorder clinically characterized by the vast, progressive loss of memory and cognition commonly in populations over the age of ~65 years, with the exception of those with familial AD, which develop AD symptoms as early as ~30 years-old. Neuropathologically, both AD and FAD can be characterized by synapse and neuronal cell loss in conjunction with accumulation of …