GENE EXPRESSION IN HUMAN KERATINOCYTES CONTAINING INTEGRATED COPIES OF SV40 EARLY REGION
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ABSTRACT GENE EXPRESSION IN HUMAN KERATINOCYTES CONTAINING INTEGRATED COPIES OF SV40 EARLY REGION By Suqing Liu Advisor: Professor Mark L. Steinberg Simian Virus 40 (SV40) large T antigen is one of the simplest and most reliable agents for induction of immortalization of cultured cells. It is known to act by blocking the activities of the cellular tumor suppressors of p53 and pRb proteins. However, other factors that may act downstream from T antigen and the mode of its regulation in SV40 transformed human keratinocytes remain unknown. In the present study we demonstrate that loss of functional T antigen results in a comprehensive gene regulation pattern that is exploited by p53 during the conversion of immortalized cells into primary cells. We employed line 130, a permanent line of SV40-immortalized human keratinocytes with a unique integrated copy of the virus to study altered gene expression after silencing of the viral early genes using RNA interference methodology. We characterized the viral integration site in 130 cells by sequence analyses of a cloned XbaI fragment isolated from a lambda bacteriophage library as well as by primer walking using various PCR techniques. The viral integrant was found to consist of two tandemly integrated viral DNA copies but with only a single intact copy of the viral early genes. The viral DNA was found to be joined, at one end, to human chromosome 21q within an intronic region of the homeobox gene, PKNOX1 and, at the other end to a site within a noncoding region of chromosome 10. The late region of the second copy adjacent to chromosome 10 was found to contain numerous rearrangements which may have occurred during the integration process that also brought about the chromosomal breakage and joining of chromosomes 21 and 10. Sequence analyses of the viral mRNAs (via cDNAs cloned in a T vector) showed that there was only a single viral transcript encoding a full length, intact early gene mRNA. SV40 large T antigen (LT) binds to the cell cycle regulators p53 and pRb, and we found that down regulation of LT antigen expression brought about an accumulation of cells at the G1/S interface, associated with cell cycle arrest. We also found increased expression of BTG2, a novel anti-proliferation protein, as well as induction of the cell cycle inhibitor p21WAF1/CIP1, murine double minute-2 promoter activity, expression of murine double minute-2 gene product and expression of GADD45A. Expression of p53 was down-regulated, but we observed an increase in p53 activity that was correlated with reduced binding to LT, suggesting that LT silencing led to p53-independent and BTG2-dependent cycle control in cell 130 line. No evidence of apoptosis was found. We attempted to establish a stable LT knock-down subline of 130 using an miRNA expression vector, but we found that while the presence of the plasmid vector was stably maintained over long-term culture, long-term LT silencing was not maintained, supporting the idea that even after long-term culture immortalization remains dependent upon SV40 T antigen expression.
Iron regulates mRNA translation initiation through RNA iron responsive element (IRE)
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The non-coding IRE-RNA structure, a 30 nt stem loop structure, regulates synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA binding with iron regulatory protein (IRP) proteins inhibits ribosome accessing mRNA. Increasing iron concentration decreases IRP binding with IRE-RNA. Previous models of IRE-mRNA translation regulation concentrate on Fe-S binding to IRP and IRP degradation after release from IRE-RNA. These models lack information on the details of decreasing IRE-RNA/IRP protein binding with iron concentration elevation. This research shows 1. Eukaryotic initiation factor 4F (eIF4F) binds to IRE-RNA with high affinity and works as a positive control element in mRNA translation. 2. eIF4F, IRP competitively bind to IRE-RNA. 3. Fe2+ increases eIF4F/IRE-RNA binding affinity, which outcompetes IRP binding. 4. Fe2+ induces an IRE-RNA conformation change leading to changes in binding affinity with eIF4F and IRP. 5. M7GTP cap doesn't affect eIF4F or IPR1 binding with 73 nt of the 5' noncoding region mRNA which includes the IRE 6. eIF4F/IRE-RNA has a much longer life time than IRP1/IRE-RNA which suggests both kinetics and stability of the complexes are important. 7. eIF4G, a subunit of eIF4F, binds to IRE-RNA without other subunits. A novel regulatory mechanism is proposed where metabolic iron (Fe2+) induces IRE-RNA conformation change to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding, indicating IRE-RNA act as a riboregulator.
MODELING SMAD DOMAINS AND THEIR INTERACTION WITH SMURF-1, C-SKI AND DNA PROMOTER MOTIF TO DESIGN INHIBITORY COMPOUNDS
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Boojala Vijay Reddy
Transforming Growth Factor-&beta (TGF-&beta) superfamily members are known for regulating wide array of cellular processes such as growth, differentiation, proliferation, and apoptosis. In the downstream of TGF-&beta signaling there are important growth and differentiation factors known as Smad proteins, which carry out the TGF-&beta responsive signaling and elicit various responses once inside the nucleus. The goal of this dissertation is texplore the available structural data of some of the molecules involved in TGF-&beta signaling process and to apply state of the art molecular modeling, docking and virtual screening tools and techniques to gain insight into the TGF-&beta signaling pathway. This study mainly concentrates on the interaction of Smad proteins with the DNA promoter motif, and other proteins c-Ski and Smurf-1 with which they interact in the signaling process. Initially MH1 domain of mammalian Smad proteins were modeled based on known crystal structure of Smad3 MH1-DNA complex (PDB ID: 1OZJ) followed by modeling of interaction pose of MH1 domain of BMP regulated Smads (Smad1/5/8) with their corresponding DNA sequence motif 5'-GCCG-3'. In this work the key residues of MH1 domain of Smad1/5/8 interacting with `GCCG' motif were identified. To investigate further the solvent accessibile contact area of key residues and binding energy calculations of modeled Smad1/5/8 MH1 with the GCCG DNA motif and GTCT DNA motif were computed. Higher free energy of binding for Smad1/5/8-MH1 complexed with nonspecific `GTCT' DNA motif compared to the GCCG motif confirmed high specificity of Smad1/5/8 with `GCCG' motif indicating that these Smads may not bind with `GTCT' DNA. Further, homology modeling approach was followed to build Smad binding domain of c-Ski, a proto-oncoprotein, which acts as co-repressor in Smad mediated TGF-&beta signaling. Various protein-protein docking methods were applied to study the interactions between the model c-Ski domain and Smad3-MH2 domain. Knowledge of biochemical data, contacts observed between key residues and solvent accessibility calculations of residues of both proteins in our top models were applied to finalize four best favored complexes of Smad3-Ski that can be used to design small molecule inhibitors antagonizing the c-Ski binding which may lead to anti-cancer drug design by appropriately regulating Smad3-Ski interaction.
Structure/Function Correlations in Pseudomonas aeruginosa DNA Ligase LigD
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The ATP-dependent DNA ligase D (LigD) performs a major role in the non-homologous end-joining (NHEJ) pathway. Pseudomonas aeruginosa LigD contains a N-terminal phosphoesterase domain (PE) domain followed by a ligase domain and a C-terminal polymerase domain. The PE domain (187 residues), belonging to a class of unique 3'- end-processing enzymes, possesses manganese dependent phosphodiesterase and phosphomonoesterase activities as it sequentially removes the 3'-ribonucleoside from the primer strand of the primer-template DNA duplex and hydrolyzes the 3'-PO4 produced finally to a 3'-OH group. Extensive mutagenesis and biochemical studies have identified critical residues and important features required for 3'- ribonuclease and 3'- phosphatase activities. Lack of sequence homology to other known nucleases lead to the belief that this enzyme possesses some unique motifs. However, in the absence of atomic level structural information clear structure/function correlations were lacking. This thesis describes the procedures used to obtain a high-resolution structure of PE domain obtained using solution NMR methods and to ascertain its interaction with DNA substrates.
Role of p53, CstF-50, and BARD1 in the Regulation of mRNA Processing following DNA Damage
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Following UV irradiation, cellular mRNA levels are transiently decreased due to the inhibition of transcription and mRNA polyadenylation. The UV-induced inhibition of 3' processing reflects the interaction of the polyadenylation factor CstF-50 with the tumor suppressors BRCA1/BARD1 as well as the BRCA1/BARD1-mediated degradation of RNA polymerase II (RNAP II). As CstF-50 can interact with BRCA1/BARD1 and RNAP II inhibiting or activating polyadenylation, respectively, and cells with reduced levels of CstF show an enhanced sensitivity to UV and reduced ability to ubiquitinate RNAP II and repair DNA, we propose a coordinating role for this factor in the DNA damage response. Our studies add complexity to the cellular response to UV treatment by linking RNA processing to the p53 network. Here, we show that the C-terminal domain of the tumor suppressor p53 associates not only with BARD1 but also with CstF-50. Although the p53/CstF complex is detected independently of DNA damage, we found that the tumor suppressor BARD1 can coexist in complexes with CstF and p53 only after UV treatment. Consistent with this, we found that the C-terminal domain of p53 inhibits pre-mRNA cleavage in vitro. Significantly, siRNA-mediated knockdown of p53 has different effects on the UV-induced inhibition of polyadenylation in cells expressing different levels of p53. Supporting the physiological significance of these results, a previously identified mutation in p53 (Ser241Phe) decreases the p53-BARD1 interaction and abrogates inhibition of polyadenylation, which can be restored by the expression of wild-type p53. We also found that p53 participates in the post-transcriptional regulation of endogenous transcripts of housekeeping but not p53-regulated genes. We also determined that the interaction between CstF-50 and BARD1 involves the ankyrin-BRCT linker but not the ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps explain the regulated assembly of different proteins involved in DNA damage to BARD1, including BARD1-dependent stabilization of p53 and the induction of apoptosis. Furthermore, supporting the role of BARD1 in signaling in the DNA damage response, we found that UV-induced BARD1 phosphorylation is important not only for its functions associated with CstF-50 but also for its functions in regulating p53 phosphorylation and stability.
Exploration of Aporphines as MDMA and AChE Inhibitors
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PART A MDMA (3,4-methylenedioxy methamphetamine) is a psychoactive drug which is thought to act via stimulation of secretion as well as inhibition of re-uptake of large amounts of serotonin, noradrenaline and dopamine in the brain. MDMA also acts directly on a number of receptors, including 5-HT2A receptors. There is considerable evidence that 5-HT2A antagonists can modulate behavioral and physiological effects of MDMA in animals. Nantenine an aporphine alkaloid ex Nandina domestica has been reported to block and reverse a range of behavioral and physiological effects of MDMA in mice. It is known that nantenine has moderate 5-HT2A antagonist activity. However, very little structure-activity relationship (SAR) studies have been performed on nantenine with regards to its activity at the 5-HT2A receptor. As part of our research focus to develop aporphine-based 5-HT2A antagonists as potential MDMA antagonists, we have prepared a library of novel analogs to investigate the structural requirements for nantenine's 5-HT2A activity. Our studies demonstrate that the N-methyl group, methylenedioxy ring and structural rigidity of the aporphine nucleus are important for activity, and that appropriate substitutions on the aromatic aporphine core can improve 5-HT2A antagonist activity. To elucidate possible binding modes of these compounds and to determine the correlation with our binding data, we built a 5-HT2A homology model based on a bovine rhodopsin template and then performed docking/scoring experiments. Our results suggest that members of the C1 series of nantenine analogs bind to the 5-HT2A receptor in the same orientation but differently than nantenine. In addition to known and important interaction between the protonated nitrogen of the ligands and Asp155 of the receptor, some of these analogs established a hydrogen bond with Ser242 as well as hydrophobic interactions with Phe234 and Gly238. These latter interactions may account for their enhanced activity as compared to nantenine. Our findings will be useful in the future design of high affinity 5-HT2A ligands based on the nantenine aporphine core structure. PART B For the second part of the project, nantenine as well as a number of flexible analogs were evaluated for acetylcholinesterase (AChE) inhibitory activity in a microplate spectrophotometric assays based on Ellman's method. It was found that the rigid aporphine core of nantenine and N-methyl substituent are important structural requirements for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors; inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ a major causative factor in the progression of Alzheimer's disease (AD).
ROLES OF IGH INTRONIC ENHANCER E&mu IN CLONAL SELECTION AT THE PRE-B TO IMMATURE B CELL TRANSITION AND IN THE ELIMINATION OF AUTOREACTIVE B CELLS
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The immunoglobulin heavy chain locus (Igh) intronic enhancer, E&mu, enhances transcription of recombined Igh genes. We have previously shown that in mice with an E&mu-deficient Igh allele (VH&deltaa), Ig&mu is expressed at half of the wild-type levels in pre-B cells. We also described an E&mu-dependent "check-point", operating at the pre-B to immature B cell transition, for heavy chain allelic exclusion. We now show that deletion of E&mu results in a smaller immature B cell compartment, and the pre-BCR/BCR signaling is diminished in pre-B cells as a result of the reduced Ig&mu levels, making it difficult for emerging BCRs to reach the signaling threshold required for positive selection of pre-B cells to the immature B cell stage. Our hypothesis is that, to circumvent the problem of inadequate signaling, E&mu-deficient B cells either 1) expand the rare precursor B cells seemingly breaking the rules of allelic exclusion to express a second IgH allele as "double-producers", to achieve higher levels of Ig&mu-chain and hence higher pre-BCR and BCR levels, or 2) undergo heightened light-chain editing to create an IgH/IgL combination with superior signaling properties to make up for the lower Ig&mu-chain (lower BCR) levels and signaling. To test these hypotheses and to determine whether escape from the developmental defects in E&mu-deficient B cells is dependent upon light chain, we provided the E&mu-deficient mice with a pre-assembled VL gene (3-83V&kappa). This led to not only a larger immature B cell compartment, but also a decrease in "double-producers". We suggest that an IgH/IgL combination with superior signaling properties may compensate for the reduced BCR levels and eliminate the selective advantage of "double-producers". We also find that "double-producers" in E&mu-deficient heterozygous mice (VH&deltaa/WTb), include a subpopulation with autoreactive BCRs. We infer that the BCRs with IgH from the VH&deltaa allele are ignored during negative selection at the pre-B to immature B cell transition, due to their low density. Instead, the double-producers are both positively and negatively selected on the basis of BCRs with IgH from the alternate allele (with E&mu intact). Taken together, these results suggest that E&mu functions to ensure sufficient Ig&mu(IgH) levels at the pre-B to immature B transition, which has an important impact on the maintenance of heavy chain allelic exclusion, the breadth of the BCR repertoire, and the elimination of autoreactive B cells.
CHARACTERIZATION OF PAXILLIN, PHOSPHOLIPASE D AND THEIR FUNCTIONAL INTERACTION
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The actin cytoskeleton plays a fundamental role in various processes including differentiation, migration, endocytosis and exocytosis. An adapter protein, paxillin, as well as an enzyme, Phospholipase D (PLD), have been associated with processes based on actin cytoskeleton regulation. Such regulation is critical for the development of Dictyostelium discoideum. To gain better insight into the roles of paxillin and PLD and to investigate their potential interactions, we study the paxillin and PLD orthologs, PaxB and PldB, respectively. Previous work showed that in Dictyostelium discoideum, paxillin (PaxB) and Phospholipase D (PldB) colocalize and co-immunoprecipitate, suggesting that they physically interact. We found that the phenotypes observed during development, cell sorting and several actin-required processes including cAMP chemotaxis, cell-substrate adhesion, actin polymerization, phagocytosis, and exocytosis reveal a genetic interaction between paxB and pldB suggesting a functional interaction between gene products. Taken together, our data point to PldB being a required binding partner of PaxB during processes involving actin eorganization. Based on our study in the model organism Dictyostelium discoideum, we examined whether a similar relationship between paxillin and PLD exists in the highly aggressive human breast cancer cell line MDA-MB-231. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK complex. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, our results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, paxillin, FAK and Erk participate in the same signaling pathway in this breast cancer cell line. The proposed studies will allow further insight into the role of these proteins in cancer and better understanding of the clinical course of disease.
SEROTONIN 1A RECEPTOR MEDIATED NEUROGENESIS IN THE DEVELOPING HIPPOCAMPUS
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The importance of the brain serotonin 1A receptor (5-HT1A-R) during postnatal brain development has been established, but the mechanism of its action in brain neurons remains unclear. It is currently known that the 5-HT1A-R plays a crucial role in the brain by regulating mood and behavior and 5-HT1A-R stimulation in adult mice has been suggested to induce neurogenesis in the adult neurogenic niches such as the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. In mice, absence of the 5-HT1A-R during development results in heightened anxiety and depressive behavior. However, the 5-HT1A-R mediated signaling activity that is responsible for its role during development is unknown. Among the hippocampal signaling pathways stimulated by the agonist-bound 5-HT1A-R, the mitogen activated protein kinase (MAPK) pathway is an important regulator of both division and survival of neuronal cells in the brain. Additionally, the Protein Kinase C (PKC) isozyme PKCɛ is an important signaling molecule that is highly expressed during early postnatal brain development particularly postnatal day 2-6 (P2-P6). Here we show that neurogenesis in the developing hippocampus at P6 in mice is dependent on both MAPK and PKCɛ. Our initial experiments use pharmacological inhibitors to confirm that PKCɛ mediates 5-HT1A-R-linked activation of MAPK. We then demonstrate that neurogenesis is increased upon stimulation of this 5-HT1A-R→PKCɛ→MAPK pathway both in a hippocampal-derived neuronal cell line stably expressing the 5-HT1A-R (HN2-5) and in the DG of P6 mice. Further, inhibition of either MAPK or PKCɛ considerably disrupts the burst in bromo-deoxy-uridine (BrdU) labeling and Ki-67 staining, showing neuroblast number, in the DG. As for a downstream signal that relays the proliferative signal from MAPK, we have identified the Retinoblastoma protein (Rb) as a potential target of MAPK, and shown that its phosphorylation dynamics may be tightly regulated in response to 5-HT1A-R stimulation. Therefore, our findings reveal a novel pathway involving PKCɛ, MAPK, and Rb through which the 5-HT1A-R potentially regulates neurogenesis during early postnatal hippocampal development.
DYNAMICS ON MULTIPLE TIMESCALES IN THE CYSTOVIRAL RNA-DIRECTED RNA POLYMERASE
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The RNA-dependent RNA polymerase P2 from cystovirus ϕ6 directs the replication and transcription of the double-stranded RNA genomes. In spite of the availability of multiple crystal structures at various points along its catalytic pathway, the dynamics role involving in the catalytic cycle and fidelity control remain unclear. Isoleucine residues are distributed over the three-dimensional fold of P2. By using the δ1 positions of 25 Ile residues as probes, we measured the dynamic modes and their alterations along P2 catalytic cycle with CPMG-based multiple quantum relaxation dispersion experiments. The results indicate that P2 is dynamic on the fast (ps-ns) and slow (μs-ms) timescale. The characteristics of several motional modes are altered in the presence of substrate analogs and single-stranded RNA templates. The timescale of the lower frequency motional modes that involve several conserved functional motifs coincides with the catalytic timescale (1-2 ms), which was determined from kinetic analyses of representative RdRPs. We further investigated the influence of the extreme 3'-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on the slow motional modes using multiple-quantum relaxation dispersion. We found that P2, in the presence of templates bearing the proper genomic 3'-ends or the preferred initiation nucleotide (GTP), displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This further suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.