Biochemistry, Biophysics, and Structural Biology Commons^™

Synthesis Of Rhamnosylated Arginine Glycopeptides And Determination Of The Glycosidic Linkage In Bacterial Elongation Factor P, Siyao Wang, Leo Corcilius, Phillip B. Sharp, Andrei Rajkovic, Michael Ibba, Benjamin L. Parker, Richard J. Payne

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

A new class of N-linked protein glycosylation – arginine rhamnosylation – has recently been discovered as a critical modification for the function of bacterial elongation factor P (EF-P). Herein, we describe the synthesis of suitably protected α- and β-rhamnosylated arginine amino acid “cassettes” that can be directly installed into rhamnosylated peptides. Preparation of a proteolytic fragment of Pseudomonas aeruginosa EF-P bearing both α- and β-rhamnosylated arginine enabled the unequivocal determination of the native glycosidic linkage to be α through 2D NMR and nano-UHPLC-tandem mass spectrometry studies.

The Complex Evolutionary History Of Aminoacyl-Trna Synthetases, Anargyros Chaliotis, Panayotis Vlastaridis, Dimitris Mossialos, Michael Ibba, Hubert D. Becker, Constantinos Stathopoulos, Grigorios D. Amoutzias

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent …

Isoacceptor Specific Characterization Of Trna Aminoacylation And Misacylation In Vivo, Kyle Mohler, Rebecca Mann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and …

Maintenance Of Transcription-Translation Coupling By Elongation Factor P, Sara Elgamal, Irina Artsimovitch, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Under conditions of tight coupling between translation and transcription, the ribosome enables synthesis of full-length mRNAs by preventing both formation of intrinsic terminator hairpins and loading of the transcription termination factor Rho. While previous studies have focused on transcription factors, we investigated the role of Escherichia coli elongation factor P (EF-P), an elongation factor required for efficient translation of mRNAs containing consecutive proline codons, in maintaining coupled translation and transcription. In the absence of EF-P, the presence of Rho utilization (rut) sites led to an ~30-fold decrease in translation of polyproline-encoding mRNAs. Coexpression of the Rho inhibitor Psu …

Design And Development Of A Plasmid Vector For Protein Expression And Purification, Mahima Grover, Craig Sweet, David H. Thompson

The Summer Undergraduate Research Fellowship (SURF) Symposium

Production and isolation of proteins are difficult, costly and time-consuming processes. The aim of this project is for the development of plasmids, which allow for streamlined production and isolation of proteins. To allow for modular insertion of varying segments of DNA we are using ‘recursive directional ligation by plasmid reconstruction’. This technique uses type II restriction endonucleases, which cut downstream from their recognition site allowing multiple insertions without losing a restriction site. Using this process, we can ligate multiple DNA sequences together and express them to be able to construct a scar less fusion protein. In order to accomplish this, …

Structural And Molecular Analysis Of A Protective Epitope Of Lyme Disease Antigen Ospa And Antibody Interactions, Shivender Shandilya, Nese Kurt Yilmaz, Ejemel Monir, Andrew Sadowski, William D. Thomas, Mark S. Klempner, Celia A. Schiffer, Yan Wang

Celia A. Schiffer

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on …

Sweetening Of Glutamine Metabolism In Cancer Cells By Rho Gtpases Through Convergence Of Multiple Oncogenic Signaling Pathways, Thambi Dorai, John T. Pinto, Arthur J. L. Cooper

NYMC Faculty Publications

Comment on: Lukey MJ, Greene KS, Erickson JW, et al. The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy. Nat Commun 2016;7:11321.

Countercurrent Chromatography Fractions Of Plant Extracts With Anti-Tuberculosis Activity, Douglas Armstrong, Nathan C. Krause, Drew Frey, J. Brent Friesen, Baojie Wan, Jordan Gunn, Scott Franzblau

Faculty Scholarship – Chemistry

Samples of numerous plant species were received from the southwestern part of the USA, from Richard Spjut, and plant samples were collected here in Illinois. All were extracted with typical solvents, giving crude residues, some of which were subjected to chromatographic methods. Some of the crude residues and some of the fractions were tested for anti-tuberculosis activity and/or antibacterial activity.

In a general way, bioactive natural products are dealt with very well by Liang & Fang. More specifically, the southwestern part of the United States has a large variety of indigenous plants many of which have not been investigated for …

Circumventing Cisplatin Resistance In Ovarian Cancers Through Reactivation Of P53 By Non-Cross-Resistant Platinum Analogs, Michelle Martinez-Rivera

Dissertations & Theses (Open Access)

Abstract

CIRCUMVENTING CISPLATIN RESISTANCE IN OVARIAN CANCERS THROUGH REACTIVATION OF P53 BY NON-CROSS-RESISTANT PLATINUM ANALOGS

Michelle Martinez-Rivera, B.S.

Advisory Professor: Zahid H. Siddik, Ph.D.

Cisplatin (cis-Pt), an anticancer platinum (Pt) drug, is used widely in the treatment of several malignancies, such as ovarian cancer. This Pt compound induces DNA damage, which results in p53 activation through post-translational modifications, mainly phosphorylation, culminating in execution of programmed cell-death. However, despite initial therapeutic response to cis-Pt, clinical resistance to this drug emerges leading to disease progression. Pt-resistance phenotypes have been associated with dysfunction in the p53 signaling pathway. Therefore, an effort to understand …

Analysis Of The Intricacies Of Substrate Recognition Of High Mobility Group Proteins And Aminoacyl-Trna Synthetases Using Non-Cognate Substrates, Douglas Van Iverson Ii

Dissertations

The studies presented in section 1 (Chapters I-IV) focus on the design and development of nucleic acid four-way junctions (4WJs) to target a member of the high mobility group (HMG) proteins, the proinflammatory cytokine high mobility group box 1 protein (HMGB1). In the present study, hybrid PNA-DNA 4WJs based on a model DNA 4WJ were constructed to improve the thermal stability of 4WJs while maintaining strong binding affinity toward HMGB1. An electrophoretic mobility shift assay (EMSA) was used to examine the binding affinity of an isolated DNA binding domain of HMGB1, the HMGB1 b-box (HMGB1b), toward a set of PNA-DNA …

The Degradation Of Pharmaceutical Pollutants In Wastewater Catalyzed By Chloroperoxidase And The Construction Of Chloroperoxidase H105r Mutant, Qinghao He

FIU Electronic Theses and Dissertations

Trace amounts of pharmaceuticals have been detected in water, from nanograms per liter to micrograms per liter, and have a negatively effect in the aquatic environment and an increased potential risk of drug poisoning for human and animals. In order to address the problem, drug degradation catalyzed by chloroperoxidase (CPO) has been investigated. CPO is a heme-containing glycoprotein secreted by the fungus, Caldariomyces fumago, it catalyzes two major types of oxidations, two one-electron oxidations as catalyzed by most peroxidases and two-electron oxidations which are rare for conventional peroxidases.

Five common drugs from a variety of classes which were persistent in …

Conformational Dynamics And Stability Associated With Magnesium Or Calcium Binding To Dream In The Regulation Of Interactions Between Dream And Dna Or Presenilins, Khoa Ngoc Pham

FIU Electronic Theses and Dissertations

Downstream regulatory element antagonist modulator (DREAM) is involved in various interactions with targets both inside and outside of the nucleus. In the cytoplasm, DREAM interacts with the C-terminal fragments of presenilins to facilitate the production of β-amyloid plaques in Alzheimer’s disease. In the nucleus, Ca²⁺ free DREAM directly binds to specific downstream regulatory elements of prodynorphin/c-fos gene to repress the gene transcription in pain modulation. These interactions are regulated by Ca²⁺ and/or Mg²⁺ association at the EF-hands in DREAM. Therefore, understanding the conformational dynamics and stability associated with Ca²⁺ and/or Mg²⁺ binding to DREAM …

Extraction, Purification And Partial Characterization Of A Carotenoid Binding Protein (Cbp) From The Epidermis Of The Monarch Butterfly Larvae (Danaus Plexippus), Nan Fang

FIU Electronic Theses and Dissertations

This dissertation describes the purification and partial characterization of CBP from the epidermis of the monarch butterfly larvae (Danaus plexippus). A yellow protein-carotenoid complex was extracted from the yellow pigmented epidermal tissue from monarch butterfly larvae by homogenization. Additional steps in the purification process included differential precipitation with ammonium sulfate, cation and anion chromatography, and lastly size exclusion chromatography. Polyacrylamide gel electrophoresis demonstrates that a single protein was isolated (M-LBP) having a ~60 kDa molecular weight, the value has subsequently been confirmed by HR-tandem MS. Lutein is the sole carotenoid bound by M-LBP with a stoichiometry of the …

Translation Control Of Swarming Proficiency In Bacillus Subtilis By 5-Amino-Pentanolylated Elongation Factor P, Andrei Rajkovic, Katherine R. Hummels, Anne Witzky, Sarah Erickson, Philip R. Gafken, Julian P. Whitelegge, Kym F. Faull, Daniel B. Kearns, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys³² of B. subtilis EF-P that is …

Multiple Quality Control Pathways Limit Non-Protein Amino Acid Use By Yeast Cytoplasmic Phenylalanyl-Trna Synthetase, Adil Moghal, Lin Hwang, Kym F. Faull, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA^Phe. We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA^Phe with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic …

Non-Canonical Roles Of Trnas And Trna Mimics In Bacterial Cell Biology, Assaf Katz, Sara Elgamal, Andrei Rajkovic, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl‐tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl‐tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond …

Quantitative Mass Spectrometry Reveals Changes In Histone H2b Variants As Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation, Matthew Rea, Tingting Jiang, Rebekah Eleazer, Meredith Eckstein, Alan G. Marshall, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here …

Zn(Ii), Cu(Ii), Sn(Ii), And Ni(Ii) And Other Metal Cations Do Not Prevent The Aggregation Of Hiapp, Charles Hoying

Honors Thesis

The Zn(II) metal ion has been shown to interact with Islet Amyloid Polypeptide (IAPP), a protein implicated in the progression of Type II Diabetes Mellitus, in such a way as to prevent the protein from aggregating into toxic fibers. We set out to find whether other metal ions might similarly prevent IAPP aggregation. Using Thioflavin T (ThT) spectroscopic assays, which measure fluorescence of ThT upon binding to aggregated IAPP, we observed a decrease in aggregation when incubated with Zn(II), Cu(II), Ni(II), and Sn(II). Atomic Force Microscopy (AFM), which can visualize fibril formation, revealed that the metals were not inhibiting IAPP …

Comparing The Quantitation Of Opiates From Possible Drug Overdose Cases Using Results Of Blood Analysis And Liver Analysis, Lee Ann Garozzo

Forensic Science Theses

Currently the quantitation of opiates at the Erie County Medical Examiner’s Office Toxicology Laboratory is conducted through whole blood analysis. The objective of this thesis project was to determine if the analysis of opiates could be conducted through liver analysis, and if the analysis of opiates would provide a more accurate quantitation compared to the blood analysis. The quantitation of opiates was conducted from the livers of sixty-four possible overdose cases that were brought into the Erie County Medical Examiner’s Office between 2013 and 2015. Results showed that the opiate drugs could successfully be quantitated using the liver analysis. Generally …

Engineering A Mutation In The Heparin Binding Pocket Of The Human Fibroblast Growth Factor, Roshni Patel

Chemistry & Biochemistry Undergraduate Honors Theses

Fibroblast growth factors (FGFs) are family of proteins that belong to a group of growth factors that are found in mammals and play an important role in angiogenesis, differentiation, organogenesis, and tissue repair. In summary, their main functionality is involved in cell division and proliferation. Because FGFs plays such a vital role in cell proliferation, they are mainly involved in the process of wound healing and injuries. FGF binds to its ligand, heparin—a heavily sulfated glycosaminoglycan. The binding of heparin to FGF occurs through electrostatic interactions, specifically between the negatively charged sulfate groups on heparin and positively charged residues such …

Characterization And Target Identification Of Ak301: A Novel Mitotic Arrest Agent, Michael J. Bond, Avijeet S. Chopra, Marina Bleiler, Michelle Yeagley, Eric Scocchera

University Scholar Projects

The Giardina Laboratory has recently identified AK301 as a novel mitotic arrest agent. This work aimed to characterize the arrest state induced by AK301 (EC₅₀ ~ 150nM) and identify the cellar targets responsible for the arrest. It was found that AK301 arrest is readily reversible upon withdrawal of AK301. Cells that slip from mitosis after removal of AK301 are sensitized to apoptosis. This was found to be unique for AK301 when compared to other mitotic arrest agents like colchicine, vincristine, and BI2536. Arrested cells were found to have increased ATM activity as well as an upregulation of p53 and …

Synthesis And Characterization Of Nanoparticle-Coupled Proteins In Human Serum Albumin, Kyle M. Mahoney

Honors College Theses

Recently, cancer has become an ever-growing issue and has led to many researchers attempt to unravel the mystery of the disease. This research has led to a promising field of treatment: nanotechnology-coupled pharmaceuticals. Nanoparticles act as a whole unit when in conjugation with other molecules and add to the carrier molecule, most often proteins, benefits the nanoparticles themselves possess. One such carrier protein that can be conjugated with nanoparticles is Human Serum Albumin (HSA). Albumin is of interest in cancer research for two reasons: it is native to the human vasculature so it does not elicit immunological reactions, and it …

Novel Compound Heterozygous Mutations Expand The Recognized Phenotypes Of Fars2-Linked Disease, Melissa A. Walker, Kyle Mohler, Kyle W. Hopkins, Derek H. Oakley, David A. Sweetser, Michael Ibba, Matthew P. Frosch, Ronald L. Thibert

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance …

Analysis Of New Hiv-1 Inhibitors As Potential Antiviral Agents For Hiv-2, Rowan Brothers

Georgia State Undergraduate Research Conference

No abstract provided.

Retigabine Holds Kv7 Channels Open And Stabilizes The Resting Potential, Aaron Corbin-Leftwich, Sayeed M. Mossadeq, Junghoon Ha, Iwona Ruchala, Audrey Han Ngoc Le, Carlos A. Villalba-Galea

School of Pharmacy Faculty Articles

The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine's action remains unknown, previous studies have identified the pore region of KV7 channels as the drug's target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 …

Role Of Flippases In Protein Glycosylation In The Endoplasmic Reticulum, Jeffrey S. Rush

Molecular and Cellular Biochemistry Faculty Publications

Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks …

Piperlongumine (Piplartine) And Analogues: Antiproliferative Microtubule-Destabilising Agents, Mary J. Meegan, Seema M. Nathwani, Brendan Twamley, Daniela M. Zisterer, Niamh O'Boyle

Articles

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and Western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerised when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, …

Synthesis And Biochemical Evaluation Of 3-Phenoxy-1,4-Diarylazetidin-2-Ones As Tubulin-Targeting Antitumor Agents, Thomas F. Greene, Shu Wang, Lisa M. Greene, Seema M. Nathwani, Jade K. Pollock, Azizah M. Malebari, Thomas Mccabe, Brendan Twamley, Niamh O'Boyle, Daniela M. Zisterer, Mary J. Meegan

Articles

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl ‘A’ ring and 4-phenyl ‘B’ ring for potent antiproliferative activity, and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC₅₀ values of 38 nM and 19 nM respectively in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular …

Probing Allosteric, Partial Inhibition Of Thrombin Using Novel Anticoagulants, Stephen S. Verespy Iii

Theses and Dissertations

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an …

Development Of A Chemical Genetic Screen To Determine Synergistic Compounds With Laromustine In Treating Glioblastoma Multiforme Cultured Cells, Ryan Weeks

Honors Theses

Laromustine is a chemotherapeutic sulfonylhydrazine prodrug used in clinical trials to treat acute myeloid leukemia (AML) and glioblastoma multiforme (GBM). While treatment of AML with laromustine has more demonstrative clinical success, there are enough promising data against GBM to pursue additional pre-clinical and clinical experiments. To determine the synergistic effects caused by treating cultured GBM cells with laromustine and a library of FDA-approved compounds, a chemical genetic screen was developed. To optimize the screen, optimal cultured GBM cell seed density, growth period and maximum well capacity were determined. The treatment period for a lethal dose of laromustine in cultured GBM …