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Amyloid fibrils have gained much attention recently within the scientific community. Amyloids are insoluble protein aggregates composed of polymerized cross-linked β sheets that are typically between 5 to 12 nm in width and have variable lengths. Amyolids have properties that make them ideal candidates for use in bionanotechnology, namely their exceptional strength, elasticity, and stability as well as their ability to self assemble. In particular, our research is interested in amyloids and their potential use in the assembly of hybrid peptide-polymer supramolecular nanostructures via noncovalent interactions. Studies are being carried out using KLVFFAE, the amyloidogenic peptide sequence of Aβ protein associated with Alzheimer’s disease. These studies focus on the effects of polyethylene glycol (PEG) modification on the amyloid self-assembly process: specifically, the modulation of nanostructures of varying sizes and configurations. Solid-phase synthesis has been used to prepare modified peptides that contain functional amino acids to enable site-specific PEGylation. Maldi-TOF was used to confirm the molecular weight of the synthesized peptides. Peptides were conjugated to PEG using carbodiimide or maleimide-thiol chemistry.
Self assembly of these conjugates into amyloid-like structures was investigated under varying temperature and shaking conditions. The resulting nanostructures were characterized using TEM and fluorescence spectroscopy.
Results indicate that the presence of PEG alters the properties of the peptide aggregates. While our initial goal is to investigate the self-assembly of hybrid bionanomaterials, we expect these efforts will contribute to a better understanding of the amyloidogenesis processes related to amyloid-related diseases and to the development of selfassembled biomaterials for various applications.
THE EFFECT OF SILVER NANOPARTICLES ON PLANT GROWTH PATTERNSCailin Jones1, Kwok-Tuen (Raymond) Tse2.
DePaul University, Chicago, IL, 2Harold Washington College, Chicago, IL.
1 Nanoparticles (NPs) are constantly consumed both voluntarily and involuntarily by living organisms and are used to make everyday products such as make up, medicines, and pesticides. Although bulk material (≥ 100 nm) and NPs ( 100 nm) are made from the same metal, NPs differ not only in size but chemical properties as well, which could pose a potential threat to an organism’s health. Metal NPs are insoluble and can easily enter a human’s body by inhalation, ingestion, or penetration through the skin. They travel through the blood stream and attach themselves to key organs. The proposed experiment entails preparing NPs and testing their effects on plant-life. It is hypothesized that silver nanoparticles (Ag-NP) will affect seed germination and growth patterns of a plant. The Ag-NPs will be
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prepared using chemical reduction to precipitate out the solid Ag-NPs. Radish and mustard-green seeds will be used for this experiment because they are easy to grow and will germinate within a short time. The seeds are cultivated in a Petri dish on a moist paper towel to observe germination rate. To analyze growth patterns, the seeds will be grown in soil and watered with water containing various concentrations of the Ag-NPs. Signs of plant abnormalities will help determine which concentration causes the most damage. The experiment with the plants has not been conducted yet, but silver nanoparticles have been successfully prepared.
CRYSTALLIZATION OF THE HETERODIMERIC 220KDA NUP84-NUP133 NUCLEAR PORE SCAFFOLDING UNITMarc Gancayco, Kevin Knockenhauer, Thomas Schwartz.
Massachusetts Institute of Technology, Cambridge, MA.
The nuclear pore complex (NPC) is a 40 to 60 MDa protein complex that coats circular openings in the nuclear envelope and serves as the main conduit for molecular transport into and out of the nucleus of the eukaryotic cell. In an effort to understand the atomic structure of the NPC, a divide-and-conquer strategy is applied. The entire NPC is a modular structure built from multiple copies of biochemically defined subassemblies that are individually amenable to structural characterization. The scaffold ring of the NPC coats the nuclear envelope and is characterized by two major subcomplexes: the heptameric Y- or Nup84-complex and the heteromeric Nic96 complex. The Y-complex has 7 universally conserved components: Nup84, Nup85, Nup120, Nup133, Nup145C, Sec13, and Seh1. Crystal structures of many Y-complex fragments are available, making it the best-characterized subcomplex of the NPC. Our work focused on solving the structure of the Nup84-Nup133 complex from yeast. The human homolog has already been characterized in part to high resolution and was used to model the interaction site for the yeast Nup84-Nup133 complex based on sequence conservation. Nup133 truncations were designed based on homology modeling to produce a minimal interacting complex ideal for crystallization. Full length Nup84 was coexpressed with truncations of Nup133 in E. coli. The complex was copurified via nickel affinity, ion exchange, and size-exclusion chromatography.
Crystallization studies are currently under way with the goal of completing a composite structure of the entire universally conserved heptameric Y-complex.
ER STRESS: A POSSIBLE MECHANISM FOR CLOZAPINE-INDUCED METABOLIC SYNDROMEUche Ozoemena1, Dina Attia1, Consuelo Walss-Bass2, Veronica Contreras-Shannon1.
St. Mary’s University, San Antonio, TX, 2University of Texas, Health Science Center at San Antonio, San Antonio, TX.
1 The atypical,antipsychotic drug clozapine causes metabolic syndrome as a side effect in some patients. The mechanism through which these side effects come about is unknown. It has been suggested that clozapine could cause mitochondrial dysfunction and endoplasmic reticulum (ER) stress, both of which are known to cause metabolic syndrome. In this study, we are exploring the effects of clozapine on ER stress. We hypothesized that treating cells with clozapine would induce ER stress. The ER is responsible for proper folding and secretion of proteins in normal cellular conditions. Under stressful conditions, however, the ER may initiate the unfolded protein response (UPR) which is intended to minimize the production of misfolded or unfolded proteins. This is accomplished by changes in gene expression and cell death in extreme cases. To test our hypothesis, we measured the expression of ER stress response genes Gadd34, Chop10, and Hspa5 in cultured mouse adipocytes (3T3-L1), myoblasts (C2C12), and monocytes (RAW 264.7) treated with increasing concentrations of clozapine: 0, 25, 50, and 75 uM, for 24 hours.
After 24 hours, RNA was isolated for qRT-PCR. In 3T3 cells, increasing concentrations of clozapine led to significant upregulation of both Chop10 and Gadd34 genes (p values of 0.0199 and 0.0097, respectively). These initial results suggest that clozapine does cause ER stress, and this may be one mechanism by which clozapine causes its adverse effects.
CHARACTERIZATION OF CHEMOTAXIS CLUSTER EXPERIMENTSErenis Lemus1, Steve Smriga2.
University of California, Santa Barbara, Santa Barbara, CA, 2Massachusetts Institute of Technology, Cambridge, MA.
1 Microscale interactions among marine microbes drive carbon and nutrient cycles in the global ocean. The ecological activities of bacteria and phytoplankton are critical to biogeochemical cycles, yet we lack a fundamental understanding of the physical behaviors that underpin the interactions between these two groups. The focus of this project will be on the physical interactions between two model organisms, the bacterium Marinobacter adherens and the diatom
SECOND GENERATION ANTIPSYCHOTIC ASENAPINE AS A POTENTIAL ADJUVANT TO CIPROFLOXACIN
THROUGH BINDING OF EFFLUX PUMP NORA IN STAPHYLOCOCCUS AUREUSStephan Kudlacek, Luis Mota-Bravo.
Biological Sciences, University of California, Irvine, Irvine, CA.
Knowledge of efflux-pump inhibition in Staphylococcus aureus by the antipsychotic drug chlorpromazine has led to testing of efflux-pump inhibition with the recently FDA approved second-generation antipsychotic asenapine (ASEN). The objective of this study is to determine the capability and potential mechanism of ASEN inhibition of NorA efflux pump activity in S. aureus. We determined the following: synergistic growth-inhibitory activity was seen with a treatment of ASEN at 50 ug/mL and 1 ug/mL of ciprofloxacin (CIP); ethidium bromide (EtBr) efflux inhibition was observed with the addition of ASEN at 50 and 100 ug/mL; growth suppression was observed with treatment of ASEN at 50 ug/mL and 2ug/mL of CIP, which led to a bacteriostatic effect after 24 hours; ASEN at 50 - 200 ug/mL with or without CIP at 2 ug/mL did not cause membrane potential perturbation; and in silico docking study revealed an ASEN binding site in a predicted NorA model. In conclusion, we propose that ASEN binds to NorA to exert synergistic effects with CIP and EtBr. This warrants further investigation of ASEN as a potential adjuvant with CIP in the management of staphylococcal infections. (Supported by NIH-MBRS-IMSD Grant GM-55246 and Minority Access to Research Careers (MARC) Program, NIH Grant GM-69337.) FRI-118
DISSECTING THE PROTEIN FEATURES THAT CONTROL SUBSTRATE SPECIFICITY OF AAA+ PROTEASESTania Gonzalez-Robles, Izarys Rivera-Rivera, Amaris Torres-Delgado, Tania A. Baker.
Massachusetts Institute of Technology, Cambridge, Cambridge, MA.
AAA+ proteases are critical in all cells as they are involved in protein quality control and in regulatory circuits. They are composed of a compartmental peptidase (e.g., ClpP14) with active sites sequestered in an interior chamber, and a hexameric ring of an AAA+ unfoldase. E. coli ClpX and ClpA are two of the best characterized AAA+ unfoldases;
these enzymes contain one or two AAA+ rings, respectively, and each has a unique N-domain that is important for substrate recognition and adaptor protein interaction. ClpS, an adaptor protein that binds the ClpA N-domain, enhances N-end rule substrate recognition and simultaneously inhibits ssrA-tagged substrate degradation. To dissect mechanisms responsible for substrate specificity, we engineered a chimeric AAA+ protease that consists of the ClpA N-domain and the AAA+ ring of ClpX (ClpANX). This chimera carries the major determinants needed for the enzyme to bind the ClpA adaptor ClpS fused to the protein-unfolding/ATPase domain of the ClpX enzyme. With this chimera, we intend to answer whether the adaptor-binding domain of ClpA is sufficient to communicate information about substrate specificity and catalytic activity to the unrelated partner enzyme ClpX. ATP hydrolysis experiments with ClpANX will show if the N-domain of ClpA affects the ATPase rate of the ClpX AAA+ ring. Fluorescent anisotropy with labeled peptide or protein substrates will allow investigation of substrate-binding affinity and specificity. Lastly, degradation experiments with differently controlled classes of substrates (e.g., N-end rule and ssrA substrates) will reveal the degree to which the N-domain rules substrate choice and delivery.
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DETERMINATION OF OXIDATION SUSCEPTIBLE CYSTEINES WITHIN MAIZE PHOSPHOENOLPYRUVATE
CARBOXYLASEFernando Vargas, Gregory Kanzaki, Scott Grover, Jamil Momand.
California State University, Los Angeles, Los Angeles, CA.
Phosphoenolpyruvate carboxylase (PEPC) is an enzyme that is critical for the fixation of carbon dioxide within C4 plants. Evidence from thiol-reactive inhibitors suggests that PEPC is regulated by reversible oxidation of the cysteine thiols. The Km of the enzyme increases 2-3 fold when treated with a thiol-reactive agent. It is not known which cysteines are responsible for altering PEPC activity. The cysteine oxidation prediction algorithm (COPA) was used to predict the PEPC cysteines that are susceptible to redox-mediated regulation. COPA predicted three sites of oxidation in PEPC (C308, C335, and C424). The goal of our experiment is to map the redox-regulated cysteines within PEPC to give us insight into mechanisms of regulation. Methoxypolyethylene glycol-maleimide (MAL-PEG), a 5000 dal thiol-reactive agent, allowed us to determine the number of cysteines per PEPC that are exposed to oxidant. Mutant forms of PEPC, where cysteines are replaced with serines, were used to map sites that are MAL-PEG reactive. Such cysteines would be predicted to be sensitive to oxidants and downregulate PEPC activity. By exposing the enzyme with a thiol-reactive agent and systematically mutating cysteines into serines, we will gain the ability to predict both the amount and site of redox-regulated cysteines within PEPC. Redox regulation at cysteine residues is a novel way to regulate PEPC. Our research will give us insight into this regulatory mechanism.
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF A UNIQUE SHELL PROTEIN FROM GLYCYL
RADICAL ENZYME BACTERIAL MICROCOMPARTMENTSHoda Ahmed, Michael Thompson, Krystal McCarty, Todd Yeates.
University of California, Los Angeles, Los Angeles, CA.
Bacterial microcompartments (MCPs) are giant protein complexes that range from 80 to 150 nm in diameter. A thin protein shell surrounds their inner contents similar to a viral capsid. Shell protein monomers of the BMC type oligomerize to form hexamers which typically bear a narrow central pore. Hexamers align tightly together forming flat surfaces that make the flat facets of the polyhedral shell. MCPs encapsulate enzymes and cofactors that are involved in specific metabolic processes. Throughout the years, MCPs have been associated with various metabolic functions.