Biochemistry, Biophysics, and Structural Biology Commons^™

Lipopolysaccharide Biosynthesis Without The Lipids: Substrate Recognition For Escherichia Coli Heptosyltransferasei, Daniel J. Czyzyk, Cassie Liu, Erika A. Taylor

Erika A. Taylor, Ph.D.

Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k(cat)/K(m) of 2.7 × 10(6) M(-1) s(-1), compared to a value of 2.2 × 10(5) M(-1) s(-1) for the Kdo(2)-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that …

Beta-Lysine Discrimination By Lysyl-Trna Synthetase, Marla S. Gilreath, Hervé Roy, Tammy J. Bullwinkle, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P is modified with (R)‐β‐lysine by the lysyl‐tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α‐ and β‐lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α‐lysine, while the G469A and A233S/G469A variants decreased stable α‐lysyl‐adenylate formation. A233S LysRS recognized β‐lysine better than wildtype, suggesting a role for this residue in discriminating α‐ and β‐amino acids. Both enantiomers of β‐lysine were substrates for tRNA aminoacylation by LysRS, which, together with …

The Trna Synthetase Paralog Poxa Modifies Elongation Factor-P With (R)-Β-Lysine, Hervé Roy, S. Betty Zou, Tammy J. Bullwinkle, Benjamin S. Wolfe, Marla S. Gilreath, Craig J. Forsyth, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non–α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of β-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

Echogenic Liposomes For Nitric Oxide Delivery And Breast Cancer Treatment, Soo Yeon Lee Female

Dissertations & Theses (Open Access)

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of …

What Is New For An Old Molecule? Systematic Review And Recommendations On The Use Of Resveratrol, Ole Vang, Nihal Ahmad, Karen Brown, Anna Csiszar, Thomas Szekeres, Thomas Walle, Joseph M. Wu

NYMC Faculty Publications

BACKGROUND: Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark.

METHODOLOGY: Literature search in databases as PUBMED and ISI Web of Science in combination with manual search was used to answer the following five questions: (1)Can resveratrol be recommended in the prevention or treatment of …

Controlling The Activity Of A Phosphatase And Tensin Homolog (Pten) By Membrane Potential, Jérôme J. Lacroix, Christian R. Halaszovich, Daniela N. Schreiber, Michael G. Leitner, Francisco Bezanilla, Dominik Oliver, Carlos A. Villalba-Galea

School of Pharmacy Faculty Articles

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor …

Chemical Tools To Characterize Membrane-Protein Binding Interactions Using Synthetic Lipid Probes, Meng Meng Rowland

Doctoral Dissertations

Signaling lipids such as diacylglycerol (DAG) and the phosphatidylinositol polyphosphates (PIP_ns) play crucial roles in numerous cellular pathways. However, characterization of their activities is hindered by the complexity of associated signaling pathways and of the membrane environment. To address this issue, we have developed lipid probes that are effective for characterizing biological events using different applications, including activity-based probing (PIP_ns and DAG) and microarray analysis (PIP_ns). The activity-based probes have been applied to label receptor targets in multiple cancer cell proteomes through photocrosslinking followed by click reactions. The probes were found to label several …

Pharmacological Chaperoning In Fabry Disease, Jerome Rogich

Masters Theses 1911 - February 2014

Fabry Disease is an X-­‐linked lysosomal storage disorder characterized by a variety of symptoms including hypohydrosis, seizures, cardiac abnormalities, skin lesions, and chronic pain. These symptoms stem from a lack of functional endogenous α-­‐ Galactosidase A (α-­GAL), which leads to an accrual of its natural substrate. The severity of the disease symptoms can be directly correlated with the amount of residual enzyme activity. It has been shown that an imino sugar, 1-deoxygalactonojirimycin (DGJ), can increase enzymatic activity and clear excess substrate. This pH-­‐dependent chaperoning phenomenon is believed to arise from the presence of aspartic acid 170 in the active site. …

Androgen Receptor-Dependent Effects Of Resveratrol On Tnsin Mrna Levels In Prostate Cancer Cells, Courtney Pisano

PCOM Biomedical Studies Student Scholarship

The chemopreventive effects of resveratrol (RSV) on cancers, including prostate cancer, have been well documented; but the mechanisms are not well known. It has been reported recently that tensin, a matrix-adhesion protein, which is greatly down-regulated in prostate cancer, has been induced by RSV in several cancer cell lines. In order to know if RSV up-regulates tensin in prostate cells, we first treated LNCaP cells with RSV and demonstrated that tensin mRNA levels were upregulated by RSV in a time and dose dependent manner. Since LNCaP cells are androgen receptor (AR) positive and previous findings have shown that RSV down-regulates …

Lead Identification Of Β-Lactam And Related Imine Inhibitors Of The Molecular Caperone Heat Shock Protein 90, Niamh O'Boyle, Andrew Js Knox, Trevor P. Price, D. Clive Williams, Daniela M. Zisterer, David G. Lloyd, Mary J. Meegan

Articles

Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the identification of inhibitors based on β-lactam and imine templates. β-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC₅₀ values of 5.6 μM, 14.5 μM and 22.1 μM respectively. The binding affinity displayed …

Preventing Mother-To-Child Transmission Of Human Immunodeficiency Virus-1 (Hiv-1): Effects Of Intrapartum And Neonatal Single-Dose Nevirapine Prophylaxis And Subsequent Hiv-1 Drug Resistance At Antiretroviral Treatment Initiation, Amanda L. Harmon

CMC Senior Theses

The prevention of mother-to-child transmission is one of the most powerful tools in human immunodeficiency virus type 1 (HIV-1) prevention and has huge potential to improve both maternal and child health. In the absence of any preventative measures, infants born to and breastfed by their HIV-positive mothers have roughly a one-in-three chance of acquiring the infection themselves. HIV can be passed on from mother-to-child during pregnancy, during labor and delivery, and even after during breastfeeding.

Intrapartum and neonatal single-dose nevirapine (sd-NVP) is the foundation of preventing mother-to-child transmission in lower resource settings where it has been used alone or as …

Synthesis, Biochemical And Molecular Modelling Studies Of Antiproliferative Azetidinones Causing Microtubule Disruption And Mitotic Catastrophe, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Niall O. Keely, Andrew Js Knox, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

Articles

The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC₅₀ value of 0.22 μM. The mechanism of action was demonstrated to be by inhibition of tubulin. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for …