Turnip Mosaic Virus Genome-Linked Protein (VPg) Inhibits Pokeweed Antiviral Protein (PAP)-Mediated Depurination of RNA
Author:
Artem Domashevskiy
Year of Dissertation:
2011
Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is a cap-binding protein, and it was suggested that it inhibits translation of RNA by binding to the 5' m7G cap structure of eukaryotic mRNA, and depurinating the mRNA at sites downstream of the cap structure. PAP is a potent antiviral agent against many plant, animal, and human viruses. Depurination of capped viral RNA may be the primary mechanism for PAP's antiviral activity. However, the above mechanism does not clarify the inhibitory effect of PAP on the replication of uncapped viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus (TuMV) genome linked protein (VPg) were investigated. VPg is important in the initiation of protein synthesis, functioning as a cap analog. VPg stimulates the in vitro translation of uncapped IRES-containing RNA and inhibits capped RNA translation in wheat germ extracts. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP-VPg interactions. PAP interacts strongly with VPg, thus the effect of VPg on the PAP catalyzed depurination of several different RNA molecules was determined to investigate whether VPg binding to PAP influences selectivity of depurination. PAP binds to and depurinates both m7GpppG-capped and uncapped S/R oligo nucleotide and TEV RNAs, supporting previous conclusions that the cap structure is not the only determinant for PAP depurination of RNA. VPg decreases depurination of the above capped and uncapped RNAs and competes with TEV RNA for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the defense mechanisms of the plant. Depurination inhibition of PAP by VPg also suggests the possible use of this protein against cytotoxic activity of RIPs and inhibition of their biological potency.
PROTEASOME FUNCTION DURING AGING IN DROSOPHILA
Year of Dissertation:
2012
Advisor:
Thomas Glenewinkel
Aging is associated with dysfunction of protein homeostasis and increased protein damage caused by oxidative stress, but the detailed molecular mechanisms are unknown. The ubiquitin-proteasome pathway (UPP) is critical to the protein quality control system as it degrades a majority of intracellular proteins in eukaryotic cells. Age-related decline in proteasome function has been shown in a variety of mammalian tissues. However the role of UPP during the aging process still remains a puzzle. In this study, we compared the UPP function between two Drosophila melanogaster strains, the longevity mutant methuselah (mth) and the wild type w1118 at different ages, which would help answer this question.
Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization
Year of Dissertation:
2011
Tight control of actin cytoskeletal dynamics is essential for proper cell function. ARNO, a guanine nucleotide exchange factor for Arf, is associated with actin cytoskeletal regulation but its exact role is unknown. To explore ARNO's role in this regulation and in actin mediated processes, the Dictyostelium discoideum homolog RnoA was examined. RnoA is involved in development, as the reduction of RnoA by antisense prolongs aggregation, delaying development. Also, RnoA overproduction arrests development at the mound stage. This arrest is rescued by the addition of wild type cells. In these chimeric mixtures, rnoA overexpressing cells (RnoA OE) preferentially sort to the stalk, suggesting RnoA plays a role in cell sorting. RnoA antisense and RnoA OE cells fail to stream during aggregation. Chemotaxis assays reveal these mutants do not chemotax toward cAMP, indicating RnoA is part of the cellular response to cAMP. This defect is specific to cAMP-directed chemotaxis, as both RnoA mutants effectively chemotax to folate and exhibit normal cell motility. The chemotactic defects of the RnoA mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. RnoA OE cells have increased filopodia compared to wild type cells, implying altered F-actin localization. Thus, RnoA likely organizes F-actin during development. Phospholipase D (PLD), the enzyme responsible for phosphatidic acid production, and paxillin, a cytoskeletal adaptor protein, are also involved in actin cytoskeletal organization. Given their communal association with the actin cytoskeleton, we explored the interactions between RnoA and the D. discoideum PLD and paxillin homologs, PldB and PaxB, respectively. PaxB and PldB regulate calcium dependent and calcium independent cell-cell cohesion, respectively. Cells lacking PaxB are known to have reduced cell-substrate adhesion. We find that overexpression of PldB rescues the adhesion defect of paxB null cells, implying a genetic interaction between paxB and pldB. Co-immunoprecipitation studies suggest RnoA, PldB, and PaxB physically interact. This interaction does not depend on PaxB, PldB, or F-actin organization. Taken together, the results suggest that RnoA, in complex with PldB and PaxB, coordinates F-actin organization during actin mediated processes such as adhesion.
New Families of Beta-Lactamase Inhibitors
Year of Dissertation:
2011
A traditional approach to improving the efficacy of â-lactam antibiotics has been to modify natural antibiotics by modifying the nucleus and adding various side chains. It is generally believed that â-lactam based antibiotics have a limited future given increased resistance demonstrated by many strains of bacteria. Therefore the lack of performance among commonly used antibiotics against the rise of resistant bacterial strains has propelled researchers to look for novel types of antimicrobial agents and enzyme inhibitors as tools to combat this serious and growing threat. The focus of this research is to investigate novel small-molecules from different families with antibacterial properties such as peptides, aromatic ketones, biphenyl and stilbene compounds and boronic acids that demonstrate an inhibitory effect on class A â-lactamase.
VEGF signaling mediates neuroprotection against oxidative stress in hippocampal neurons in vivo and in vitro
Year of Dissertation:
2013
Advisor:
Patricia Rockwell
Vascular endothelia growth factor (VEGF) signaling through its cognate receptor VEGFR-2 has been shown to be neuroprotective against stressful stimuli including oxidative stress. Using a rat pilocarpine model of status epilepticus (SE), pilocarpine-induced SE was associated with the induction of the oxidative stress marker Heme oxygenase-1 (HO-1) and increased expression of the pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). Treatments with exogenous VEGF attenuated the induction in HO-1 and the increase in COX-2 levels. Previous studies showed that VEGF protected hippocampal cell death in this model and these results suggest that oxidative stress and inflammation contribute to this neuronal loss. Similarly, in cultured hippocampal neurons, pilocarpine induced neuronal cell death accompanied with increased HO-1 expression, reactive oxygen species (ROS) production, caspase-3 cleavage and a loss of mitochondrial membrane potential (ÄØm) that were all suppressed by VEGF. To delineate the protective mechanisms associated with VEGF-mediated neuroprotection, VEGFR-2 inhibition was investigated with respect to oxidative stress in primary cultures of hippocampal neurons. Treatments with the pharmacological inhibitor SU1498 elicited a cytotoxicity that was prevented by the antioxidant N-acetyl-cysteine (NAC) and accompanied by induction of HO-1, cleavage of caspase-3, production of ROS together with a loss of ÄØm. Knockdown of VEGFR-2 by siRNA generated a similar pattern of ROS and ÄØm loss. Treatments with VEGF or VEGF-B prevented neurons from the cell death and mitochondrial dysfunction induced by SU1498 or siRNA, suggesting that a molecular switch occurs where both growth factors may signal through VEGFR-1 when VEGFR-2 activity is blocked. Consistent with a general role for pro-apoptotic BAD and anti-apoptotic Bcl-xL in neuronal cell death and oxidative stress, the phosphorylation of MEK/ERK1/2 and BAD (inactivation) and the protein levels of Bcl-xL that were attenuated by SU1498 were somewhat increased by VEGF. Our findings support a role for VEGF in signaling BAD inactivation and increased Bcl-xL as a specific mechanism that protects hippocampal neurons against oxidative stress. These findings also suggest that VEGF activates VEGFR-2 to protect neurons against oxidative stress but can signal through alternate receptors to serve the same function.
Isolation and Characterization of Immunomodulatory Compounds from Juzen-Taiho-To: Novel Understanding of Phytosteryl Glucosides Nano-Aggregates and Synergism
Year of Dissertation:
2009
This thesis describes the realization of the immunomodulatory properties of four phytosteryl-glucosides betta;-sitosterol-D-Glucoside, campesteryl betta-D-glucoside, 24-epicampestryl betta-D-glucoside, Stigmasteryl betta-D-glucoside) purified from a Japanese Kampo herbal formulation termed Juzen-Taiho-To (JTT). JTT has been used for almost a thousand years and currently is prescribed to people with an impaired immune system. Our screening methodology termed Genomic-Screening allowed us to investigate the immunomodulatory properties of JTT in a new and exciting way. Genomic Screening employs the use of GeneChip® oligonucleotide array, and quantitative reverse-transcriptase polymerase chain reaction qRT-PCR. THP-1 human cancerous monocytic cell line was used in our study with JTT due to their accurate representation of healthy monocytes as well as a large variety of receptors. THP-1 treatment with JTT revealed many genes to be differentially regulated most of which were down stream elements of NFkappa B pathway. We chose (ICAM-1) for our screening since it was the most reproducible assay and it also had a high fold change. By monitoring ICAM-1 response to JTT treatment we were able to uncover known phytosteryl-glucosides that previously were not known for their immunostimulatory properties. In addition our study discovered that a specific ratio of our phytosteryl glucosides that synergistically induced ICAM-1 expression. We have also discovered that the purification approach of the four phytosteryl glucosides greatly affects the immunostimulatory properties. Using Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) we were able to show that nano-aggregated of betta-sitosterol-D-Glucoside formed in a water containing solution diminished ICAM-1 expression in THP-1 cells. These findings underline the importance of natural product chemistry as well as the approach to purifying small organic molecule from natural resources. The underlining mechanism of how our phytosteryl glucosides induce an immune response is still unknown. Nevertheless our finding has opened the door to new research avenues with a family of compound that contain a novel class of pathogen associated molecular patterns.
Role of Fibroblast Growth Factor Homologous Factors in Excitability of Hippocampal Neurons
Year of Dissertation:
2009
Advisor:
Mitchell Goldfarb
Fibroblast growth factor homologous factors (FHFs) are a family of vertebrate neuronal proteins which function in manners distinct from fibroblast growth factors (FGFs). While bearing substantial sequence and structural homology to FGFs, FHFs reside intracellularly and bind targets unrelated to FGF receptors, which include voltage-gated sodium channels (Navs). FHFs have been shown to control excitability of cerebellar granule cells through modulation of channel inactivation. Since action potentials are initiated at axon initial segment (AIS), we suspected that at least some FHF isoforms would reside at AIS in association with Navs. As expected, a broad repertoire of FHF isoforms colocalizes with Navs at the AIS of cultured hippocampal neurons. Moreover, together with other studies in our laboratory, the present study shows that FHF "b" isoform associates with AIS to a far lesser extent than "a" isoform, demonstrating that there is isoform specificity in FHF targeting. AIS targeting of an FHF requires the protein's channel binding surface, as a mutant derivative of FHF2a deficient for channel binding is also deficient in AIS targeting.
PROTEASOME IMPAIRMENT, MITOCHONDRIA DYSFUNCTION, AND TAU PATHOLOGY IN ALZHEIMER DISEASE
Year of Dissertation:
2012
Advisor:
MARIA FIGUEIREDO-PEREIRA
Impairment of the ubiquitin/proteasome pathway is implicated in the pathogenesis of many neurodegenerative disorders, such as Alzheimer disease (AD). This is supported by detection of ubiquitinated protein aggregates in neurofibrillary tangles (NFTs) (Ross and Poirier, 2004) and reduction of proteasome activity in autopsied brains from AD patients (Keller et al., 2000). However, the upstream events leading to impairment of the ubiquitin/proteasome pathway are not fully understood. The ubiquitin/proteasome pathway is a highly regulated and efficient pathway that is critical for degradation of most intracellular proteins (Ciechanover, 2005). Ubiquitination, proteasome assembly and proteasome activity are energy dependent processes that require ATP binding and ATP hydrolysis (Li and Demartino, 2009). In neurons, most ATP is generated by mitochondria. Emerging evidence implicates mitochondrial dysfunction and energy deficit in many neurodegenerative disorders such as AD (Lin and Beal, 2006).
Uncovering hidden potential of natural products
Year of Dissertation:
2010
Herbs and soil bacteria have been arguably the most important sources of therapeutic agents throughout human history. Numerous bioactive compounds have been isolated and characterized from these classical sources of natural products. Because of the long history of research on herbs and soil bacteria, they are sometimes perceived as “exhausted” sources of secondary metabolites. These classical sources of natural products, however, can still include compounds with previously overlooked chemical and biological properties. The unifying theme of this thesis study is to uncover such hidden potential of natural products. To this end, these classical sources of secondary metabolites have been examined from new angles.
Peptide Directed 3D Assembly of Nanoparticles through Biomolecular Interaction
Year of Dissertation:
2012
The current challenge of the `bottom up' process is the programmed self-assembly of nanoscale building blocks into complex and larger-scale superstructures with unique properties that can be integrated as components in solar cells, microelectronics, meta materials, catalysis, and sensors. Recent trends in the complexity of device design demand the fabrication of three-dimensional (3D) superstructures from multi-nanomaterial components in precise configurations. Bio mimetic assembly is an emerging technique for building hybrid materials because living organisms are efficient, inexpensive, and environmentally benign material generators, allowing low temperature fabrication.