Molecular Biology Commons^™

An Interdomain Sector Mediating Allostery In Hsp70 Molecular Chaperones, Robert G. Smock, Olivier Rivoire, William P. Russ, Joanna F. Swain, Stanislas Leibler, Rama Ranganathan, Lila Gierasch

Lila Gierasch

Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector’, which is an attribute of the allosteric Hsp70 sub-family that links the …

Role Of Hsp70 Atpase Domain Intrinsic Dynamics And Sequence Evolution In Enabling Its Functional Interactions With Nefs, Ying Liu, Lila Gierasch, Ivet Bahar

Lila Gierasch

12 Hide Figures Abstract Author Summary Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic …

Aromatic Acid Metabolites Of Escherichia Coli K-12 Can Induce The Marrab Operon, Lon Chubiz, Christopher Rao

Biology Department Faculty Works

MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity.

Frataxin And Mitochondrial Fes Cluster Biogenesis, Timothy L. Stemmler, Emmanuel Lesuisse, Debumar Pain, Andrew Dancis

Biochemistry and Molecular Biology Faculty Publications

Friedreich’s ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in Fe-S cluster assembly in mitochondria. Fe-S clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multi-step and multi-subunit mitochondrial machinery that includes a scaffold protein Isu for assembling a protein bound Fe-S cluster intermediate. Frataxin interacts with Isu, iron, and with the cysteine desulfurase Nfs1 that supplies sulfur, thus placing it at the center of mitochondrial Fe-S cluster biosynthesis.

Bilinear Programming And Protein Structure Alignment, J. Cain, D. Kamenetsky, N. Lavine

Mathematical Sciences Technical Reports (MSTR)

Proteins are a primary functional component of organic life, and understanding their function is integral to many areas of research in biochemistry. The three-dimensional structure of a protein largely determines this function. Protein structure alignment compares the structure of a protein with known function to that of a protein with unknown function. A protein’s three-dimensional structure can be transformed through a smooth piecewise-linear sigmoid function to a real symmetric contact matrix that represents the functional significance of certain parts of the protein. We address the protein alignment problem as a minimization of the 2-norm difference of two proteins’ contact matrices. …

Role And Regulation Of Epha2 In Pancreatic Cancer, Pavel A. Levin

Dissertations & Theses (Open Access)

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets.

The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found …

Characterizing The Role Of Dna Repair Proteins In Telomere Length Regulation And Maintenance: Fanconi Anemia Complementation Group C Protein And 8-Oxoguanine Dna Glycosylase, David Beomjin Rhee

Doctoral Dissertations

Telomeres are the chromosome end structures consisting of telomere-associated proteins and short tandem repeat sequences, TTAGGG, in humans and mice. Telomeres prevent chromosome termini from being recognized as broken DNA ends. The structural integrity of DNA including telomeres is constantly threatened by a variety of DNA damaging agents on a daily basis. To counteract the constant threats from DNA damage, organisms have developed a number of DNA repair pathways to ensure that the integrity of genome remains intact. A number of DNA repair proteins localize to telomeres and contribute to telomere maintenance; however, it is still unclear as to what …

Characterization Of Regulatory Mechanisms For Alternative Splicing In Alpha Thyroid Hormone Receptor Mrna, Chao Zhang

Master's Theses (2009 -)

In eukaryotes, alternative splicing is an essential post transcriptional modification process for functional gene expression and a major contributor to protein diversity. The regulation of alternative splicing generally involves the engagements of RNA sequences cis-acting elements) and corresponding protein factors (trans-acting factors). The cisacting RNA motifs can be categorized depending on positional and functional differences. Trans-acting protein factors will then bind to RNA sequences and affect the corresponding splicing activity. Recently, factors associated with 3’ polyadenylation have also been identified to affect alternative splicing.

In mammals, the α-thyroid hormone receptor gene (TRα) produces transcripts for two functionally antagonistic isoforms, TRα1 …

The Role Of Car And Pxr In Toxicant Sensitivity, Linda Mota

All Dissertations

The Constitutive Androstane Receptor (CAR) and the Pregnane X Receptor (PXR) are nuclear receptors of significant importance in the regulation of enzymes that metabolize, detoxify and eliminate compounds from the body. In this study we assessed the protective role of CAR and PXR in the basal and inducible regulation of Cytrochrome P450s (CYPs), and the potential of CAR and PXR to help protect individuals from the organophosphate, parathion and the plasticizer, nonylphenol, putatively due to improved metabolism and elimination. Knockout models of these receptors were used to model susceptible populations such as children that are known to have lower CAR …

Specific, Reversible Cytostatic Protection Of Normal Cells Against Negative Effects Of Chemotherapy, Benjamin B. Mull

Dissertations & Theses (Open Access)

Chemotherapy is a common and effective method to treat many forms of cancer. However, treatment of cancer with chemotherapy has severe side effects which often limit the doses of therapy administered. Because some cancer chemotherapeutics target proliferating cells and tissues, all dividing cells, whether normal or tumor, are affected. Cell culture studies have demonstrated that UCN-01 is able to reversibly and selectively arrest normal dividing cells; tumor cells lines do not undergo this temporary arrest. Following UCN-01 treatment, normal cells displayed a 50-fold increase in IC50 for camptothecin; tumor cells showed no such increased tolerance.

We have examined the response …

Artemis Interacts With The Cul4a Ubiquitin E3 Ligase Complex And Regulates The Cell Cycle Progression, Yiyi Yan

Dissertations & Theses (Open Access)

Artemis, a member of the SNM1 gene family, is one of the six known components of the non-homologous end joining pathway. It is a multifunctional phospho-protein that has been shown to be modified by the phosphatidylinositol 3-kinases (PIKs) DNA-PKcs, ATM and ATR in response to a variety of cellular stresses. Artemis has important roles in V(D)J recombination, DNA double strand breaks repair and damage-induced cell-cycle checkpoint regulation. The detailed mechanism by which Artemis mediates its functions in these cellular pathways needs to be further elucidated. My work presented here demonstrates a new function for Artemis in cell cycle regulation as …

Fosinopril, A Potential Substrate For Mrp2, Competes With Several High Use Pharmaceuticals For Elimination, Benjamin Green

All Theses

The multidrug-resistance associated protein 2 (MRP2) is a membrane-bound transporter responsible for the efflux of a variety of drugs and endogenous compounds. MDCK cells transfected with the human MRP2 gene were used to assess whether several highly used pharmaceuticals were potential substrates by examining their differential toxicity, accumulation, and efflux. Fosinopril, an ACE inhibitor, was 2.4-fold less toxic to the MRP2 transfected cells compared to mock transfected cells, suggesting that fosinopril is a potential MRP2 substrate. In addition, fosinopril was effluxed more rapidly, as the MRP2 cells only retained 13 % of the dosed fosinopril after 20 minutes compared with …

Four Aspects Of Dogwood Pollination: Insect Visitation, A Novel Approach To Identify Pollen, Floral Volatile Emission, And Tracking Parentage, Paul Raymond Rhoades

Masters Theses

Part 1: Visitation of potential pollinators to Cornus florida and C. kousa flowers was assessed in East Tennessee in 2008 and 2009. Data regarding insect visitation rates to multiple trees per location were gathered throughout the flowering period. Diurnal and seasonal variations in visitation were recorded. Pollen coverage was assessed on portions of captured insect exoskeletons that were most likely to contact the stigma. Eleven families in four insect orders were collected from C. florida and 26 families in five orders from C. kousa. The most important pollinators in eastern Tennessee were bees in the Andrenidae and Halictidae. The most …

The Larval Salivary Gland Of Drosophila Melangogaster: A Model System For Temporal And Spatial Steroid Hormone Regulation, Benjamin Constantino

UNLV Theses, Dissertations, Professional Papers, and Capstones

Drosophila melanogaster provides an ideal model organism to test genetic and molecular biological mechanisms within the context of a living animal. For over one hundred years Drosophila continues to produce a boundless extent of informative and important scientific data providing crucial insight into development, disease progression and genetic interactions. A century as a model organism allowed for the development of an abundance of unique genetic and molecular tools allowing researchers to tease apart cellular mechanisms with very little limitation. From the whole adult body to tissue function to molecular networks, if a biological question arises it most likely can be …

Poxa, Yjek And Elongation Factor P Coordinately Modulate Virulence And Drug Resistance In Salmonella Enterica, William Wiley Navarre, Shicong Zou, Hervé Roy, Jinglin Lucy Xie, Alexei Savchenko, Alexander Singer, Elena Edvokimova, Lynne R. Prost, Runjun Kumar, Michael Ibba, Ferric C. Fang

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-β-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered expression of multiple proteins, including several encoded on the SPI-1 pathogenicity island. PoxA mediates posttranslational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homolog, eIF5A, with …

Release Of Hmgb1 In Response To Pro-Apoptotic Glioma Killing Strategies: Efficacy And Neurotoxicity, Marianela Candolfi, Kader Yagiz, David Foulad, Gabrielle Alzadeh, Matthew Tesarfreund, Akm Ghulam Muhammad, Mariana Puntel, Kurt Kroeger, Chunyan Liu, Sharon Lee, James Curtin, Gwendalyn D. King, Jonathan Lerner, Katsuaki Sato, Yohei Mineharu, Weidong Xiong, Pedro R. Lowenstein, Maria Castro

Articles

Purpose In preparation for a Phase I clinical trial utilizing a combined cytotoxic/immunotherapeutic strategy using adenoviruses expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor killing agent in relation to efficacy and safety when compared to other pro-apoptotic approaches. Experimental Design and Results The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the pro-apoptotic cytokines (TNF-α, TRAIL, FasL), alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. In rats bearing large GBMs (day 9), the …

Rna Processing Of Nitrogenase Transcripts In The Cyanobacterium Anabaena Variabilis, Justin Ungerer, Brenda Pratte, Teresa Thiel

Biology Department Faculty Works

Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, …

Rna Processing Of Nitrogenase Transcripts In The Cyanobacterium Anabaena Variabilis, Justin L. Ungerer, Brenda S. Pratte, Teresa Thiel

Teresa Thiel

Redox Status Affects The Catalytic Activity Of Glutamyl-Trna Synthetase, Assaf Katz, Ranat Banerjee, Merly De Armas, Michael Ibba, Omar Orellana

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were …

Molecular Basis For Distinct Pathways For Protein Import Into Arabidopsis Chloroplasts, Danny Schnell, C. Rounds, H. Inoue

Danny Schnell

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate …

Notch1 Functions As A Tumor Suppressor In A Model Of K-Ras–Induced Pancreatic Ductal Adenocarcinoma, Linda Hanlon, Jacqueline L Avila, Renée M Demarest, Scott Troutman, Megan Allen, Francesca Ratti, Anil K Rustgi, Ben Z Stanger, Fred Radtke, Volkan Adsay, Fenella Long, Anthony J Capobianco, Joseph L Kissil

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased …

Growth Hormone Alters Components Related To Differentiation, Metabolism And Milk Synthesis And Secretion In Mac-T Cells, Tasha Lynn Johnson

Master's Theses

The mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin and is extensively used to study bovine mammary epithelial cell function. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and induce cytoskeletal rearrangement in 3T3 fibroblast cell line and a Chinese hamster ovary (CHO) cell line. Few studies have focused on identifying the mechanisms involved in differentiated MAC-T cells’ response to GH. We tested the hypothesis that MAC-T cells would respond directly to GH and that …

Mimosa: A System For Minimotif Annotation, Jay Vyas, Ronald J. Nowling, Thomas Meusburger, David P. Sargeant, Krishna Kadaveru, Michael R. Gryk, Vamsi Kundeti, Sanguthevar Rajasekaran, Martin Schiller

Life Sciences Faculty Research

BACKGROUND:

Minimotifs are short peptide sequences within one protein, which are recognized by other proteins or molecules. While there are now several minimotif databases, they are incomplete. There are reports of many minimotifs in the primary literature, which have yet to be annotated, while entirely novel minimotifs continue to be published on a weekly basis. Our recently proposed function and sequence syntax for minimotifs enables us to build a general tool that will facilitate structured annotation and management of minimotif data from the biomedical literature.

RESULTS:

We have built the MimoSA application for minimotif annotation. The application supports management of …

Intrinsic Contact Geometry Of Protein Dynamics, Yosi Shibberu, Allen Holder, David Cooper

Mathematical Sciences Technical Reports (MSTR)

We introduce a new measure for comparing protein structures that is especially applicable to analysis of molecular dynamics simulation results. The new measure generalizes the widely used root-mean-squared-deviation (RMSD) measure from three dimensional to n-dimensional Euclidean space, where n equals the number of atoms in the protein molecule. The new measure shows that despite significant fluctuations in the three dimensional geometry of the estrogen receptor protein, the protein's intrinsic contact geometry is remarkably stable over nanosecond time scales. The new measure also identifies significant structural changes missed by RMSD for a residue that plays a key biological role in …

Dopaminergic Innervation Of The Subventricular Zone In The Murine Brain, Linda Beth Drozdowicz

Honors Scholar Theses

The subventricular zone (SVZ) is one of two areas in the brain that, in a healthy mouse, continually generate neurons throughout adulthood. While it was previously thought that only the A9 neurons of the substantia nigra sent dopaminergic afferents to the SVZ, recent studies suggest that the A10 neurons of the ventral tegmental area may innervate this area. This project has aimed to discover which, if either, model is correct.

Examination of the Aphakia (AK) mouse was used to determine the role of distinct midbrain regions in SVZ regulation. Additionally, intraperitoneal injections of the chemical MPTP were used to deduce …

Characterization Of The Putative Xyloglucan Glycosyltransferase Gt14 In Arabidopsis Thaliana, Najam R. Syed

Honors Scholar Theses

Plant cell walls largely consist of matrix polysaccharides that are linked to cellulose microfibrils. Xyloglucan, the primary hemicellulose of the cell wall matrix, consists of a repeating glucose tetramer structure with xylose residues attached to the first three units ('XXXG'). In Arabidopsis thaliana, the core XXXG structure is further modified by enzymatic addition of galactose and fucose residues to the xylose side chains to produce XLXG, XXLG, XLLG and XLFG structures. GT14 is a putative glycosyltransferase in the GT47 gene family. Initial predictions of GT14's hydrophobic regions, based on its translated amino acid sequence, are almost identical to its Arabidopsis …

Alkylphenol Contamination In Homarus Americanus, Jennifer Renee Urban

Honors Scholar Theses

Alkylphenols are pollutants that are present in marine sediments and fishes. In earlier work it has been discovered that alkylphenols are present in the Homarus americanus, or the American lobster. Research suggests that alkylphenols could behave as endocrine disruptors as they have been found to affect juvenile hormone activity. It has been hypothesized that lobsters may be able to rid themselves of alkylphenol contamination through secreting these compounds into the environment or sequestering them in their tissues. In this study, I address the question of how lobsters may rid themselves of alkylphenols by analyzing hemolymph, muscle, gill, and shell samples …

Determination Of The Myogenic Potential Of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells, Rory Coleman

Honors Scholar Theses

Human embryonic stem cells (hESCs) have the potential to

differentiate to all adult somatic cells. This property makes hESCs a very promising area of research for the treatment of disorders in which specific cell populations need to be restored. Despite this potential, research that focuses on producing mesodermally derived cell populations from hESCs is decidedly limited, notwithstanding the prevalence of disorders involving mesodermal tissues for which treatment options are limited. Skeletal muscle myoblasts are derivatives of mesodermal cells and are characterized by the expression of the MyoD gene. These cells are difficult to obtain from hESCs in a reproducible and …

Dynamics Of The Toc Gtpases: Modulation By Nucleotides And Transit Peptides Reveal A Mechanism For Chloroplast Protein Import, Lovett Evan Reddick

Doctoral Dissertations

The chloroplast is the green organelle in the plant cell responsible for harvesting energy from sunlight and converting it into sugars and ATP. Origins of this organelle can be traced back to an endosymbiotic event in which a primitive eukaryotic cell capable of oxidative phosphorylation engulfed a free-living cyanobacterium capable of photosynthetic respiration (1). Immediately following this event the details are not clear, however what is known is that over the course of evolution, the engulfed cyanobacteria relinquished approximately 97% of its protein coding sequences to the host cell nucleus, thus making the newly formed chloroplast reliant on its host …

Functional Analysis Of Chromodomain Helicase Dna Binding Protein 2(Chd2) Mediated Genomic Stability, Sangeetha Rajagopalan

Doctoral Dissertations

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. …