Biochemistry, Biophysics, and Structural Biology Commons^™

Carbachol Regulation Of Akt In Lncap Prostate Cancer Cells, John M. Schmitt, Luke Fletcher

Faculty Publications - Department of Biological & Molecular Science

Abstract Hormones and agonists that enhance cell survival through binding specific G Protein-Coupled Receptors (GPCRs) are of particular interest in cancer cell survival. LNCaP cells have been shown to express muscarinic cholinergic receptors that are responsive to the agonist, carbachol. In addition, LNCaP cells specifically express the M3-subtype of GPCR’s that may couple carbachol to Gq, increases in intracellular calcium, and activation of the intracelluar Ca2+/calmodulin-dependent protein kinases (CaM Ks). The CaM Kinase family of proteins includes CaM KII, CaM Kinase Kinase (CaM KK) and its substrates CaM KIV, CaM KI and the protein kinase AKT. AKT is an anti-apoptotic …

Plant-Like Substitutions In The Large-Subunit Carboxy Terminus Of Chlamydomonas Rubisco Increase Co2/O2 Specificity, Sriram Satagopan, Robert Spreitzer

Department of Biochemistry: Faculty Publications

Background: Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is ~90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes.
Results: The quadruple-mutant enzyme has a 10% increase in CO₂/O₂ specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to …

Structural Basis Of The Transcriptional Regulation Of The Proline Utilization Regulon By Multifunctional Puta, Yuzhen Zhou, John D. Larson, Christopher A. Bottoms, Emilia C. Arturo, Michael T. Henzl, Jermaine L. Jenkins, Jay C. Nix, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon–helix–helix domain (PutA52, a polypeptide corresponding to residues 1–52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gelshift analysis. Three of the sites are shown to be …

Comparative Genomic Analyses Of Copper Transporters And Cuproproteomes Reveal Evolutionary Dynamics Of Copper Utilization And Its Link To Oxygen, Perry Ridge, Yan Zhang, Vadim N. Gladyshev

Vadim Gladyshev Publications

Copper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. …

Identification Of A Consensus Dna-Binding Site For The Arabidopsis Thaliana Sbp Domain Transcription Factor, Atspl14, And Binding Kinetics By Surface Plasmon Resonance, Xinwen Liang, Tara J. Nazarenus, Julie M. Stone

Department of Biochemistry: Faculty Publications

Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized …

A Functional Link Between Housekeeping Selenoproteins And Phase Ii Enzymes, Aniruddha Sengupta, Bradley A. Carlson, James A. Weaver, Sergey V. Novoselov, Dmitri E. Fomenko, Vadim N. Gladyshev, Dolph Hatfield

Vadim Gladyshev Publications

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II …

Reduced Reliance On The Trace Element Selenium During Evolution Of Mammals, Alexey Lobanov, Dolph Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Background: Selenium (Se) is an essential trace element that occurs in proteins in the form of selenocysteine (Sec). It is transported throughout the body in the form of Sec residues in Selenoprotein P (SelP), a plasma protein of unclear origin recently proposed as an experimental marker of dietary Se status.
Results: Here, we report that the amino-terminal domain of SelP is distantly related to ancestral bacterial thiol oxidoreductases of the thioredoxin superfamily, and that its carboxy-terminal Se transport domain may have originated in early metazoan evolution by de novo accumulation of Sec residues. Reconstruction of evolutionary changes in the Se …

In Silico Identification Of Genes Involved In Selenium Metabolism: Evidence For A Third Selenium Utilization Trait, Yan Zhang, Anton Turanov, Dolph Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Background: Selenium (Se) is a trace element that occurs in proteins in the form of selenocysteine (Sec) and in tRNAs in the form of selenouridine (SeU). Selenophosphate synthetase (SelD) is required for both utilization traits. However, previous research also revealed SelDs in two organisms lacking Sec and SeU, suggesting a possible additional use of Se that is dependent on SelD.
Results: In this study, we conducted comparative genomics and phylogenetic analyses to characterize genes involved in Se utilization. Candidate genes identified included SelA/SelB and YbbB that define Sec and SeU pathways, respectively, and NADH oxidoreductase that is predicted to generate …

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 …

History Of The Department Of Biochemistry And Molecular Biology, Gene A. Homandberg

UND Departmental Histories

This departmental history was written on the occasion of the UND Quasquicentennial in 2008.

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 …

Characterizing The Dynamics And Functional Role Of Site-Specific Phosphorylation Of G Protein-Coupled Receptors, John M. Busillo

Full-Text Theses & Dissertations

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) and the subsequent recruitment of arrestin is a well-established paradigm that initiates the process known as desensitization. However, an emerging theme in GPCR regulation is the possibility of differential regulation dictated by the phosphorylation pattern elicited by the different members of the GRK family. Therefore, we have used small interfering RNA-mediated knock down of the GRKs and arrestins in an attempt to better understand how phosphorylation regulates the activity and signaling of the M3 muscarinic acetylcholine receptor (M3 mAChR) and CXCR4, two receptors endogenously expressed in HEK293 cells.

Using a …

Urine Protein Electrophoresis: Comparison Of Agarose Gel And Capillary Zone Electrophoretic Techniques., Anne-Marie Curtin

Theses

Proteinuria is the excess excretion of protein in the urine and is found in a variety of conditions including renal diseases and neoplastic conditions such as multiple myeloma (MM). The investigation of proteinuria type through the use of electrophoresis is a useful and widely used non-invasive diagnostic tool. The aim of this study was to compare various agarose gel and capillary zone electrophoresis techniques in order to establish a method choice for the separation of protein in routine laboratories. Comparisons were made based on urine monoclonal peak quantification and protein fractions detected. An examination of a capillary zone electrophoresis (CZE) …

Development Of Srap, Srap-Rga, Rapd, And Scar Markers Linked With A Fusarium Wilt Resistance Gene In Eggplant, Nedim Mutlu, Filiz Hatice Boyacı, Münevver Göçmen, Kazım Abak

Department of Biochemistry: Faculty Publications

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melon­gena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F2 and BC1 populations of eggplant segregating for …

Naturally Present Fatty Acids As Internal Calibrants For Fourier Transform Mass Spectra Of Dissolved Organic Matter, Rachel L. Sleighter, Georgina A. Mckee, Zhanfei Liu, Patrick G. Hatcher

Chemistry & Biochemistry Faculty Publications

The analysis of dissolved organic matter ( DOM) by Fourier transform ion cyclotron resonance mass spectrometry ( FTICR- MS) has gained wide interest recently, driven primarily by its ultrahigh resolving power and mass accuracy. Accurate calibration of mass spectra is a key step to successfully decipher the DOM components. We propose a simple and accurate method to internally calibrate the peaks in the complex spectra without the need to add a calibrant. Mass spectra of DOM samples from the Dismal Swamp, Virginia, and the lower Chesapeake Bay display the presence of naturally occurring fatty acids which can be readily recognized …

Functional Domains Of The Fatty Acid Transport Proteins: Studies Using Protein Chimeras, Concetta C. Dirusso, Dina Darwis, Thomas Obermeyer, Paul N. Black

Department of Biochemistry: Faculty Publications

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of …

The Human Hyaluronan Receptor For Endocytosis (Hare/Stabilin-2) Is A Systemic Clearance Receptor For Heparin, Ed Harris, Janet A. Weigel, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K_d for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and …

7Β-Hydroxy­Artemisinin, Paulo B. Carvalho, Bo Liu, Yunshan Wu, John S. Williamson, Mitchell A. Avery

Office for Research Publications and Presentations

Crystals of the title compound [systematic name: (3R,6R,7S,8aR,9R,12aR)-7-hydr­oxy-3,6,9-trimethyl­octa­hydro-3,12-ep­oxy[1,2]dioxepino[4,3-i]isochromen-10(3H)-one], C₁₅H₂₂O₆, were obtained from microbial transformation of artemisinin by a culture of Cunninghamella elegans. The stereochemistry of the compound is consistent with the spectroscopic findings in previously published works. A weak O—H⋯O hydrogen bond occurs in the crystal structure, together with intermolecular C—H⋯O hydrogen bonds.

Transcriptional Regulation Of Lamb3 By P53, Meghna Jani

Browse all Theses and Dissertations

The p53 tumor-suppressor plays a very important role in the prevention of cancer and it is known that about 50% of all human tumors possess p53 mutations. Although mutations in p53 are most prevalent in human cancers, inactivation of wild-type p53 occurs through many different mechanisms that are independent of p53 mutation or deletion. In an effort to determine novel p53 target genes, our lab employed a microarray method in which p53 was re-activated by RNAi mediated knockdown of Hdm2 and HdmX in MCF7 human breast cancer cell line, harboring wild-type p53 and elevated levels of Hdm2 and HdmX. Gene …

Ancient Dna Identification Of Early 20th Century Simian T-Cell Leukemia Virus Type 1, Sebastien Calvignac, Jean-Michel Terme, Shannon M. Hensley, Pierre Jalinot, Alex D. Greenwood, Catherine Hanni

Biological Sciences Faculty Publications

The molecular identification of proviruses from ancient tissues (and particularly from bones) remains a contentious issue. It can be expected that the copy number of proviruses will be low, which magnifies the risk of contamination with retroviruses from exogenous sources. To assess the feasibility of paleoretrovirological studies, we attempted to identify proviruses from early 20th century bones of museum specimens while following a strict ancient DNA methodology. Simian T-cell leukemia virus type 1 sequences were successfully obtained and authenticated from a Chlorocebus pygerythrus specimen. This represents the first clear evidence that it will be possible to use museum specimens to …

M3-Muscarinic Receptor Activation Of Erk And Cell Growth Requires Calcium/Calmodulin-Dependent Protein Kinases In Mcf-7 Cells, John M. Schmitt, Ellen Abell, Andrea Wagner

Faculty Publications - Department of Biological & Molecular Science

Abstract The extracellular signal-regulated protein kinase (ERK) signaling pathway is found in diverse cells throughout the human body. ERK activation has been implicated in breast cancer cell growth and proliferation. Studies have shown that ERK is activated by carbachol, a G Protein-Coupled Receptor (GPCR) agonist, which increases intracellular calcium in MCF-7 cells. The calcium/calmodulin-dependent protein kinase (CaM K) family of proteins including CaM KK, CaM KI, and CaM KII can be activated by increased intracellular calcium. Our goal was to determine whether CaM Ks may be responsible for ERK activation and cell proliferation in carbachol-treated MCF-7 cells and evaluate which …

Chiral Recognition Of Amino Acids By Use Of A Fluorescent Resorcinarene, Gerald I. Richard, Hadi M. Marwani, Shan Jiang, Sayo O. Fakayode, Mark Lowry, Robert M. Strongin, Isiah M. Warner

Chemistry Faculty Publications and Presentations

The spectroscopic properties of a chiral boronic acid based resorcinarene macrocycle employed for chiral analysis were investigated. Specifically, the emission and excitation characteristics of tetraarylboronate resorcinarene macrocycle (TBRM) and its quantum yield were evaluated. The chiral selector TBRM was investigated as a chiral reagent for the enantiomeric discrimination of amino acids using steady-state fluorescence spectroscopy. Chiral recognition of amino acids in the presence of the macrocycle was based on diastereomeric complexes. Results demonstrated that TBRM had better chiral discrimination ability for lysine as compared to the other amino acids. Partial least squares regression modeling (PLS-1) of spectral data for macrocycle-lysine …

Identification Of Lactic Acid Bacteria In Michigan Cherry Wines, Emily Henk, Margaret Dietrich, Terri Weese

Student Summer Scholars Manuscripts

Many factors contribute to the final flavor of wine. One factor is malolactic fermentation, during which lactic acid bacteria (LAB) transform the harsh tasting malic acid into a more drinkable lactic acid in grape wine. The role of LAB in the production of cherry wine is completely unknown. The goal of this study is to identify the species of LAB in cherry wine and compare them to those found in grape wines. Bacteria from cherry wine were grown on general media plates and plates fortified with malic acid, which may provide optimal growing conditions for the LAB. To identify the …

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 …

Validation Of A Custom-Made Microarray To Study Human Intestinal Microflora, Harshavardhan Kenche

Browse all Theses and Dissertations

Intestinal microflora refers to all the different species of bacteria that reside in the human gut and is an important organ of the human body because almost all the digestive reactions of the host occur in the intestine. The bacteria of the intestine play a key role in this process by supplementing the intestine with various enzymes and proteins that are required for the digestive process. At the same time, these bacteria were shown to be implicated in a variety of gastrointestinal disorders like Irritable Bowel Syndrome, Inflammatory Bowel Disorder and Gastrointestinal Cancer, but with the current knowledge about the …

Development Of An Automated Method For Csf Bilirubin Measurement., Helen Caverley

Theses

Subarachnoid haemorrhage (SAH) is a potentially fatal condition which can be difficult to accurately diagnose. Patients often present with a second bleed which can be more damaging than the initial bleed. Diagnosis of SAH centres on computed tomography (CT) scans. However, 2% of patients positive for SAH will present as negative on CT scans. It is recommended that these patients have lumbar puncture and the CSF is analysed for bilirubin. The presence of bilirubin is suggestive of SAH. Spectrophotometric scanning involves expensive equipment which is often not available in routine settings. A need exists for a robust, inexpensive, automated method …