Biochemistry, Biophysics, and Structural Biology Commons^™

Studying Potential Drug Interactions Involved In The Regulation Of The Diaphanous-Related Formins, Brittany E. Benson, Bradley J. Wallar

Student Summer Scholars Manuscripts

Diaphanous-related formins (DRFs) are a conserved family of proteins that are involved in the regulation of cellular shape, motility, and cell division by regulating the structure of the cellular “skeleton” (cytoskeleton); any disruption of this regulation can result in cell death. Normally, the DRFs are kept in an inactive state by the intramolecular binding of two regions of the protein: the Diaphanous Autoregulatory Domain (DAD) and Diaphanous Inhibitory Domain (DID). This binding can be alleviated by various naturally-occurring mechanisms and proper regulation of the activity of these proteins is vital to cell survival because prolonged activation of DRFs can result …

The Role Of Protein Disulfide Isomerase (Pdi) In Oxidative Folding, Veronica Gonzalez

Open Access Theses & Dissertations

Protein Disulfide Isomerase is a highly evolved oxidoreductase enzyme that has the capability of oxidizing, reducing, and isomerizing disulfides in substrate. PDI is organized into several domains denoted a, b, b', a' and c. These domains are believed to have different functions but all must be present for full PDI activity. In this manuscript we recorded the ability of PDI b' to catalyze oxidative folding in fully reduced RNase A. We also examined competition between thiol-disulfide shuffling and conformational folding by PDI. This competition creates a rough effect of a highly efficient enzyme in two different substrates: RNase A and …

Involvement Of Inflammation In Tau Pathology And Mechanisms That Drive Neurodegeneration In Alzheimer's Disease, Lisette T. Arnaud

Dissertations, Theses, and Capstone Projects

Two of the hallmarks of neurodegenerative disorders, such as Alzheimer's disease (AD), are (1) the appearance of proteinaceous deposits in inclusion bodies containing aggregates of ubiquitinated proteins and (2) activated microglia and astrocytes surrounding the diseased neurons. In Alzheimer's disease, the intracellular inclusion bodies are known as neurofibrilary tangles (NFT). The mechanisms leading to inclusion body formation and their role in the progression of neurodegeneration are still largely unknown.

Many of the proteins that accumulate in inclusion bodies depend on the ubiquitin/proteasome pathway (UPP) for their degradation. This pathway is responsible for the bulk (∼80%) of intracellular protein degradation. Because …

Mapping The Functional Interaction Of Sco1 And Cox2 In Cytochrome Oxidase Biogenesis, Kevin Rigby, Paul A. Cobine, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Sco1 is implicated in the copper metallation of the Cu_A site in

Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated

and apo-conformers revealed structural dynamics primarily

in an exposed region designated loop 8. The structural

dynamics of loop 8 in Sco1 suggests it may be an interface for

interactions with Cox17, the Cu(I) donor and/or Cox2. A series

of conserved residues in the sequence motif ²¹⁷KKYRVYF²²³ on

the leading edge of this loop are shown presently to be important

for yeast Sco1 function. Cells harboring Y219D, R220D, V221D,

and Y222D mutant Sco1 proteins failed …

Function And Redox State Of Mitochondrial Localized Cysteine-Rich Proteins Important In The Assembly Of Cytochrome C Oxidase, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The cytochrome c oxidase (CcO) complex of the mitochondrial respiratory chain exists within the mitochondrial inner membrane (IM). The biogenesis of the complex is a multi-faceted process requiring multiple assembly factors that function on both faces of the IM. Formation of the two copper centers of CcO occurs within the intermembrane space (IMS) and is dependent on assembly factors with critical cysteinyl thiolates. Two classes of assembly factors exist, one group being soluble IMS proteins and the second class being proteins tethered to the IM. A common motif in the soluble assembly factors is a duplicated Cx₉C sequence …

Pet191 Is A Cytochrome C Oxidase Assembly Factor In Saccharomyces Cerevisiae, Oleh Khalimonchuk, Kevin Rigby, Megan Bestwick, Fabien Pierrel, Paul A. Cobine, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The twin-Cx₉C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx₉C motif class of proteins in being independent of the Mia40 pathway.

Coa2 Is An Assembly Factor For Yeast Cytochrome C Oxidase Biogenesis That Facilitates The Maturation Of Cox1, Fabien Pierrel, Oleh Khalimonchuk, Paul A. Cobine, Megan Bestwick, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is dependent on a new assembly factor designated Coa2. Coa2 was identified from its ability to suppress the respiratory deficiency of coa1 Δ and shy1 Δ cells. Coa1 and Shy1 function at an early step in maturation of the Cox1 subunit of CcO. Coa2 functions downstream of the Mss51-Coa1 step in Cox1 maturation and likely concurrent with the Shy1-related heme a₃ insertion into Cox1. Coa2 interacts with Shy1. Cells lacking Coa2 show a rapid degradation of newly synthesized Cox1. Rapid Cox1 proteolysis also occurs in shy1 Δ cells, suggesting …

Purification From Human Milk Of Matriptase Complexes With Secreted Serpins: Mechanism For Inhibition Of Matriptase Other Than Hai-1, I-Chu Tseng, Feng-Pai Chou, Sheng-Feng Su, Michael Oberst, Nandakumar Madayiputhiya, Ming-Shyue Lee, Jehng-Kang Wang, David E. Sloane, Michael Johnson, Chen-Yong Lin

Department of Biochemistry: Faculty Publications

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages …

Degenerate Codon Mutagenesis And Cloning Of G-Protein Coupled Receptor Gene Fusion, Michael J. Sofolarides

Seton Hall University Dissertations and Theses (ETDs)

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Electrical Detection Of The Temperature Induced Melting Transition Of A Dna Hairpin Covalently Attached To Gold Interdigitated Microelectrodes, Greg P. Brewood, Yaswanth Rangineni, Daniel J. Fish, Ashwini Bhandiwad, David R. Evans, Raj Solanki, Albert S. Benight

Chemistry Faculty Publications and Presentations

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5' end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25 degrees C to 80 degrees C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded …

Novel Functions Of Acyl-Coa Thioesterases And Acyltransferases As Auxiliary Enzymes In Peroxisomal Lipid Metabolism., Mary Hunt, Stefan Alexson

Articles

Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells, and to date have been shown to contain approximately 60 identified enzymes involved in various metabolic pathways, including the oxidation of a variety of lipids. These lipids include very long-chain fatty acids, methyl branched fatty acids, prostaglandins, bile acid precursors, and xenobiotics that are either β-oxidized or α-oxidized in peroxisomes. The recent identification of several acyl-CoA thioesterases and acyltransferases in peroxisomes has revealed their various functions in acting as auxiliary enzymes in α- and β-oxidation in this organelle. To date, 9 functional acyl-CoA thioesterases and acyltransferases have been …

The Nudix Hydrolase 7 Is An Acyl-Coa Diphosphatase Involved In Regulating Peroxisomal Coenzyme A Homeostasis., Sarah-Jayne Reilly, Veronica Tillander, Rob Ofman, Stefan Alexson, Mary Hunt

Articles

Coenzyme A (CoASH) is an obligate cofactor for lipids undergoing β-oxidation in peroxisomes. Although the peroxisomal membrane appears to be impermeable to CoASH, peroxisomes contain their own pool of CoASH. It is believed that CoASH enters peroxisomes as acyl-CoAs, but it is not known how this pool is regulated. The mouse nudix hydrolase 7 (NUDT7α) was previously identified in peroxisomes as a CoAdiphosphatase, and therefore suggested to be involved in regulation of peroxisomal CoASH levels. Here we show that mouse NUDT7α mainly acts as an acyl-CoA diphosphatase, with highest activity towards medium chain acyl-CoAs, and much lower activity with CoASH. …

Preparation And Nucleophilic Substitution Of The 2,4,6-Triphenylpyridinium Salts, Diazonium Intermediates And N,N-1,2-Benzenedisulfonylimides Of Chiral Amino Acids, S. A. Said

Tanzania Journal of Science

Three methods for preparation of D-amino acids by nucleophilic substitution on derivatives of their L-antipodes have been attempted. Nucleophilic substitution on optically active phenylalanine ethyl ester triphenylpyridinium salt yielded partial inverted azide products. The N,N-disulfonylimide intermediate of the same ester was synthesized in high yield and optical purity, but the substitution reaction on this compound was not achieved. Diazotization of amino acid ammonium chlorides and tosylates in solution of sodium azide in aprotic solvents gave optically active chloro and tosyl compounds instead of the intended azide products. Alternative methods to circumvent the problem of the competing ammonium salt counterions duringdiazotization …

Preparation Of Divalent And Trivalent Antigens For Immunogical Studies On Degranulation Of Mast Cells And Preparation Of Ceragenins For Antiviral Studies Against Vaccinia Virus., Dianliang Geng

Theses and Dissertations

CHAPTER 1

Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that …

Pyridinium Bisretinoids: Synthesis And Photoactivated Cytotoxicity, Junping Gao

Theses and Dissertations

This thesis discusses pyridinium bisretinoid compounds (PBRs), which were prepared for two purposes: 1) to use them as standards for detection of novel fluorophores in human RPE cells, which may be involved in age-related macular degeneration (AMD), and 2) to use them in the development of a targeted and triggered drug delivery system for cancer therapy. We prepared a selection of PBRs using a one-pot biomimetic method; synthesis, mechanisms for formation, and characterization of these compounds is described. We also explored the photoreactivity of three novel PBR compounds and found that these PBRs form oxidation products under blue-light irradiation. The …

Study Of The Reproducibility Of Proteomics Methods And Variability Of Fruit Fly Proteomes., Thomas Franklin Culwell

Theses and Dissertations

The reliability of biomarker discovery by means of proteomics has been called into question. It was speculated that "background noise" variation resulting from differences in preparation and handling of samples and proteome dynamics may mask subtle, yet important, differences due to the biological condition. Little is understood about complex proteomes and their variability. A critical aspect of proteomic biomarker research that is largely unexplored is the comparative reproducibility of certain methods such as two-dimensional gel electrophoresis and liquid chromatography/mass spectrometry. In particular, with liquid chromatography/mass spectrometry, it is not known whether variability in peptide quantitation is dependent on any of …

Pyridinium Bis-Retinoids A2-Dopamine And A2-Cadaverine: Implications In Age-Related Macular Degeneration And Cancer, Mckenzie Ruth Pew

Theses and Dissertations

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer …

Studies In The Biochemistry And Cell Biology Of Trypanosoma Brucei Hexokinases, Jeremy Chambers

All Dissertations

Trypanosoma brucei expresses two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 did not, a finding attributed to differences in the C-termini of the proteins. We found that the C-terminal tails of TbHK contained a novel motif specific for eukaryotic hexokinases found within the final helix of an ATP-binding domain. By altering the residues in the C-terminal tail of TbHK1 to resemble the residues found in TbHK2, we found that D454, F462, M466, and N469 were essential to TbHK1 catalysis. Additionally, we found …

Effects Of Water Vapor On The Kinetics Of The Methylperoxy Radical Self-Reaction And Reaction With Hydroperoxy, Alecia Mower

Theses and Dissertations

The gas phase reactions of CH3O2 + CH3O2, HO2 + HO2, and CH3O2 + HO2 in the presence of water vapor have been studied at temperatures between 263 and 303 K using laser flash photolysis coupled with UV time-resolved absorption detection at 220 nm and 260 nm. Water vapor concentration was quantified using tunable diode laser spectroscopy operating in the mid-IR. The HO2 self-reaction rate constant is significantly enhanced by water vapor, consistent with what others have reported, whereas CH3O2 self-reaction and the cross-reaction (CH3O2 + HO2) rate constants are nearly unaffected. The enhancement in the HO2 self-reaction rate coefficient …

The Tropical Forest And Fire Emissions Experiment: Method Evaluation Of Volatile Organic Compound Emissions Measured By Ptr-Ms, Ftir, And Gc From Tropical Biomass Burning, T. G. Karl, Ted J. Christian, Robert J. Yokelson, Paulo Artaxo, Wei Min Hao, Alex Guenther

Chemistry and Biochemistry Faculty Publications

Volatile Organic Compound (VOC) emissions from fires in tropical forest fuels were quantified using Proton-Transfer-Reaction Mass Spectrometry (PTRMS), Fourier Transform Infrared Spectroscopy (FTIR) and gas chromatography (GC) coupled to PTRMS (GC-PTR-MS). We investigated VOC emissions from 19 controlled laboratory fires at the USFS (United States Forest Service) Fire Sciences Laboratory and 16 fires during an intensive airborne field campaign during the peak of the burning season in Brazil in 2004. The VOC emissions were dominated by oxygenated VOCs (OVOC) (OVOC/NMHC similar to 4:1, NMHC: non-methane hydrocarbons) The specificity of the PTR-MS instrument, which measures the mass to charge ratio of …

1,3,7,10,14,17,21,28,31,42,52,55-Dodeca­Kis(Trifluoro­Meth­Yl)- 1,3,7,10,14,17,21,28,31,42,52,55-Dodeca­Hydro­(C60-IH)[5,6]Fullerene, Natalia B. Shustova Prof. Dr., O.P. Anderson, O. V. Boltalina, S. H. Strauss, I. E. Kareev

Faculty Publications

The title compound, C₇₂F₃₆, is one of four isomers of C₆₀(CF₃)₁₂ for which crystal structures have been obtained. The fullerene mol­ecule has an idealized I_h C₆₀ core with the 12 CF₃ groups arranged in an asymmetric fashion on two ribbons of edge-sharing C₆(CF₃)₂ hexa­gons, a para–meta–para–para–para–meta–para ribbon and a para–meta–para ribbon, giving an overall pmp ³ mp,pmp structure. There are no cage Csp ^{3 …}

Microchip Liquid Chromatography And Capillary Electrophoresis Separations In Multilayer Microdevices, Hernan Vicente Fuentes

Theses and Dissertations

In this dissertation, several microfabricated devices are introduced to develop new applications in the area of chemical analysis. Electrochemical micropumps, chip-based liquid chromatography systems and multilayer capillary electrophoresis microdevices with crossover channels were fabricated using various substrates such as poly(dimethylsiloxane) (PDMS), glass, and poly(methyl methacrylate) (PMMA). I have demonstrated pressure-driven pumping of liquids in microfabricated channels using electrochemical actuation. PDMS-based micropumps were integrated easily with channel-containing PMMA substrates. Flow rates on the order of ~10 µL/min were achieved using low voltages (10 V). The potential of electrolysis-based pumping in microchannels was further evaluated for pressure driven microchip liquid chromatography (LC). …

Microfluidic Electro-Osmotic Flow Pumps, John Mason Edwards

Theses and Dissertations

The need for miniaturized, portable devices to separate and detect unknown compounds has greatly multiplied, leading to an increased interest in microfluidics. Total integration of the detector and pump are necessary to decrease the overall size of the microfluidic device. Using previously developed thin film technologies, an electroosmotic flow (EOF) pump was incorporated in a microfluidic liquid chromatography device. An EOF pump was chosen because of its simple design and small size. EOF pumps fabricated on silicon and glass substrates were evaluated. The experimental flow rates were 0.19-2.30 microliters/minute for 40-400 V. The theoretical pump efficiency was calculated along with …

Discovery Of Platelet-Type 12-Human Lipoxygenase Selective Inhibitors By High-Throughput Screening Of Structurally Diverse Libraries., Joshua D. Deschamps, Jeffrey T. Gautschi, Stephanie Whitman, Tyler A. Johnson, Nadine C. Gassner, Phillip Crews, Theodore R. Holman

Natural Sciences and Mathematics | Faculty Scholarship

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors …

Counter-Flow Ion Mobility Analysis: Design, Instrumentation, And Characterization, Nosa Agbonkonkon

Theses and Dissertations

The quest to achieve high resolution in ion mobility spectrometry (IMS) has continued to challenge scientist and engineers in the field of separation science. The low resolution presently attainable in IMS has continued to negatively impact its utility and acceptance. Until now, efforts to improve the resolution have mainly focused on better instrumentation and detection methods. However, since the resolution of IMS is diffusion limited, it makes sense to address this limitation in order to attain high resolution. This dissertation presents a new IMS technique, which utilizes a high electric field and opposing high gas flow velocity with the aim …

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which …

Optimization Of Non-Natural Nucleotides For Selective Incorporation Opposite Damaged Dna, Diana Vineyard, Xuemei Zhang, Alison Donnelley, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the π-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of …

Gene Synthesis, Cloning, Expression, Purification And Biophysical Characterization Of The C2 Domain Of Human Tensin, Kiran Sukumar Gajula

Doctoral Dissertations

Tensin is a large "docking" protein found in the adhesive junctions of animal cells and recruited early in the development of cell-substrate contacts. There it binds to the cytoplasmic domain of integrin β1 and caps the barbed ends of filamentous actin. This forms a rational basis for its implication in a direct role in the mechanics of membrane-cytoskeleton interactions. Tensin provides a physical link between the actin cytoskeleton, integrins, and other proteins at the cell-substrate contacts. Its overall biochemical properties are a function of its domain composition and architecture, i.e., the domains that are present and their relative positions in …

A Simple Array Platform For Microrna Analysis And Its Application In Mouse Tissues, Xiaoqing Tang, Jozsef Gal, Xun Zhuang, Wang-Xia Wang, Haining Zhu, Guiliang Tang

Plant and Soil Sciences Faculty Publications

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes. Most miRNAs are conserved between humans and mice, which makes it possible to analyze their expressions with a set of selected array probes. Here, we report a simple array platform that can detect 553 nonredundant miRNAs encompassing the entire set of miRNAs for humans and mice. The platform features carefully selected and designed probes with optimized hybridization parameters. Potential cross-reaction between mature miRNAs and their precursors was investigated. The array platform was …

High-Throughput Screening For Fatty Acid Uptake Inhibitors In Humanized Yeast Identifies Atypical Antipsychotic Drugs That Cause Dyslipidemias, Hong Li, Paul N. Black, Aalap Chokshi, Angel Sandoval-Alvarez, Ravi Vatsyayan, Whitney Sealls, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12). The library used consisted of 2,080 compounds with known biological activities. Of these, ~1.8% reduced cell-associated C1-BODIPY-C12 fluorescence and were selected as potential inhibitors of human FATP2- mediated fatty acid …