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Strategic targeting of curcumin to eliminate brain tumors
Year of Dissertation:
Glioblastoma, the most common form of primary brain cancer, is highly aggressive and associated with very poor prognosis. Curcumin (or diferuloylmethane), a natural molecule, which is not toxic to normal tissue, has been shown to inhibit proliferation, induce apoptosis and inhibit angiogenesis and metastasis in a wide range of cancer cells. However, the effective delivery of curcumin to cancers presents a problem because curcumin is poorly soluble in water and metabolizes quickly. Our preliminary work has established that solubilized curcumin can cross the blood-brain barrier and is harmless to normal brain cells, that solubilized curcumin blocks brain tumor formation when introduced by injection into the blood or directly into the brain, and that it markedly decreases cell viability in several cell lines, including murine melanoma B16F10 and murine glioblastoma GL261. Targeted drug delivery is frequently used to deliver drugs selectively and at high concentrations to cancer tissue. Antibody-mediated targeting, in addition to delivering drugs selectively, serves to increase the water solubility of attached drugs. We postulated that antibody-mediated targeting would be an effective means of eliminating brain tumors. Nonetheless, a number of structural features had to be carefully considered. Curcumin has several functional groups, which potentially can be used to target the molecule to cancer cells; however, curcumin's functional groups have been shown to be critical for its anticancer activity. With this in mind, after weighing different options, we synthetically modified curcumin at its phenolic hydroxyl position to enable the formation of a cleavable antibody attachment. Intracellular hydrolysis of an ester bond returns curcumin to its original state after its delivery into target cells. Murine infiltrating melanoma (B16F10) and primary glioblastoma (GL261) brain tumor models were utilized. We created two adducts, curcumin-MUC18 for targeting to B16F10 cells and curcumin-CD68 for targeting to GL261 cells. Our studies show that both adducts are highly effective at eliminating B16F10 and GL261 cancer cells in vitro, and that these adducts destroy cancer cells at far lower concentrations than does free curcumin. Our molecular analyses show that, in GL261 cells, curcumin causes a dramatic increase in caspase 3/7 activity and suppression of tumor-promoting proteins NF-κB, Akt1, VEGF, cyclin D1, and BclXL. We show in GL261 cells that overexpressed NF-κB is protective against curcumin treatment. Lastly, we show that animals implanted with B16F10 or GL261 cells receiving targeted curcumin treatment live longer and have significantly reduced tumor size.
POTENTIAL ROLE OF ATP8A1 AS PLASMA MEMBRANE AMINOPHOSPHOLIPID TRANSLOCASE IN PROLIFERATING NEURONAL CELLS
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The inner leaflet-localized phospholipid PS undergoes a translocation to the outer leaflet of the plasma membrane in apoptotic cells to trigger recognition and phagocytic removal of these dying cells by PS receptor-bearing scavenger cells, such as microglia and macrophages. The enzyme activity responsible for the inner-membrane localization of PS is the plasma membrane aminophospholipid translocase (PLAPLT), also known as flippase, which translocates PS from the outer to the inner leaflet of the plasma membrane. Attempts to identify the PLAPLT molecule of mammalian cells have revealed a candidate molecule, Atp8a1, which is a P-type Mg-ATPase. After much controversy, it is currently believed that Atp8a1 translocates PS across internal membranes but not the plasma membrane. Based on our earlier studies showing overexpression of Atp8a1 in proliferating hybrid neuroblastoma cells causes an increase PLAPLT activity, we postulated that Atp8a1 functions as PLAPLT only in fast dividing cells, such as neurotumor cells or neuroblasts. This study used the fluorescent PS analogue, NBD-PS, to show that ectopic expression of Atp8a1 in the N18 neurotumor cells causes no significant change in the Km value of PLAPLT, but an increase in the Vmax for this enzyme, which suggests that overexpression of Atp8a1 causes an increase in the PLAPLT molecules. This indicates that Atp8a1 is possibly identical to the PLAPLT molecule of the N18 cells. As a confirmation of this hypothesis we expressed phosphorylation-site mutants of Atp8a1 in the N18 cells to elicit a decrease in the Vmax value of PLAPLT, without significantly altering the Km value. The inhibition of the PLAPLT activity in N18 cells was also evidenced by a striking increase in surface staining of these cells with the PS-binding protein annexin V. According to our postulate Atp8a1 deletion should also cause PS exposure in neuroblasts harbored within the dentate gyrus (DG), which is a proliferative niche within the memory center termed hippocampus. In corroboration, we observed pronounced annexin V staining in both dissociated DG cells as well as cultured hippocampal slices of Atp8a1 (-/-) mice but not wild type mice. Such PS externalization should trigger phagocytosis of DG cells, which in turn could lead to a loss of hippocampal function. In support of this postulate we have observed that the Atp8a1 (-/-) mice suffer from possibly hippocampal-related learning defects. Therefore, Atp8a1 may play a crucial role in the maintenance of the functional integrity of the hippocampus. Additionally, our study reveals a potential strategy for the selective removal of the brain tumor cells through targeted suppression of Atp8a1 activity in brain cancer cells, which would lead to PS externalization and elimination of the cells by phagocytosis.
Conformational Dynamics of Guanine Residues Within the Human Telomeric G-quadruplex
Year of Dissertation:
The human telomeric single-stranded guanine-rich DNA (HT4: d(TTAGGG)4) located at the end of chromosomes forms an intramolecular G-quadruplex in the presence of K+ or Na+ in vitro. The formation and stabilization of this structure by quadruplex interactiveagents (QIAs) can inhibit the activity of the enzyme complex telomerase, which is overactivated in cancer cells, and is thus a target for potential cancer therapeutics. However, the solution quadruplex conformation is complex and varies with the presence of stabilizing Na+ or K+-ions. In addition, details about the contribution of individual guanine residues of the single stranded HT4 sequence required for G-quadruplex formation and stabilization remain unclear. In our studies, we have focused on the conformational dynamics of the human telomeric HT4 intramolecular G-quadruplex in solution, specifically the role each guanine residue plays in the quadruplex folding process, through investigating their contributions to the global quadruplex stabilities and the local environmental changes around single guanine residue. To accomplish this aim, we have substituted single guanine residues with the fluorescence analog-6MI at guanine positions (G1, G2, G4, G5, G7, G9, G11 and G12) along the HT4 oligonucleotide. Serving as either both a base mutation and a sensitive probe for local environment changes, the 6MI-substituted oligonucleotides have provided insights into the quadruplex dynamics under physiological conditions. We have confirmed the formation of G-quadruplex conformations in the presence of 100mM KCl or NaCl by all 6MI-substituted sequences using thermal difference spectroscopy. In general, the global stabilities of the 6MI-labeled sequences (as judged by mid-point of thermal UV-melting profiles and &Delta Gfolding are weaker than for the parent HT4, but to varying extents, suggesting that different guanine positions of the quadruplex do indeed play distinct roles in the formation and stabilization of the G-quadruplex. In the presence of K+, mutations at guanine positions near the 5' and 3'-ends (G2, G4, G9, G11 and G12) destabilize the quadruplex more severely than those positions at the middle (G5 and G7) of the HT4, due to the better accommodation of bulky 6MI at G5 and G7 through bulging out. For the Na+-promoted quadruplex conformation, however, the center tetrad guanine mutations appear to have the greatest impact on global stabilities, suggesting their critical roles in chelating with the cations for quadruplex stability. Studies of local environmental effects around individual guanine positions have been explored using the sensitivity of 6MI fluorescence. With quadruplex folding promoted through either decreasing temperatures or addition of K+, a decrease in fluorescence intensity generally corresponds to formation of quadruplex. The degree of fluorescence quenching however, varied for different 6MI-labeled sequences, suggesting different extents of base stacking interactions associated with quadruplex folding for each guanine residue. Analyses of fluorescence data from thermal folding or K+ titration studies suggested a cooperative quadruplex folding pathway, with base nucleation starting at relatively low K+-ion concentrations (<20mM). In the K+-stabilized quadruplex, nucleation initiates around the center tetrad (G5 and G11), followed by loop formations (G4, G7 and G9), and finally folding of the terminal ends (G1, G2 and G12). In contrast, for Na+-stabilized quadruplex folding, the top G-tetrad (G1, G7 and G12) appears to initiate folding, followed by guanines on the middle G-tetrad (G2, G5 and G11). Interestingly, the G1 labeled position (close to the 5'-end) shows abnormal behavior compared with other substituted positions on folding, with a fluorescence intensity enhancement and spectral shifts to longer wavelength. Our data suggests the flexibility of G1 on the 5'-end of the sequence in the folded quadruplex conformation with possible strand fraying and H-bonding interactions, over base stacking interactions, as predicted for all other guanine positions. Time-resolved fluorescence studies were performed to address possible mechanisms for the observed steady-state fluorescence quenching with quadruplex folding. Fluorescence intensity decay profiles were best fit using three decay components for all 6MI-labeled sequences. Although static quenching is evident, presumably arising from base stacking interactions, the observed intensity quenching is dominated by an ultrafast quasi-static self- quenching deactivation route (QSSQ). This effect is greatest for 6MI positions G5 and G11 sandwiched by two guanines, and lowest for G1 near the 5'-end. On K+-initiated folding, an increase in QSSQ is detected, suggesting that the additional fluorescence intensity quenchingmay arise from additional (sub-picosecond) electron transfer events or base stacking interactions around the 6MI probe. Fluorescence decay-associated spectra (DAS), which associate lifetimes (or decay rates) with fluorescence spectral envelopes, can provide insights into the origins of the heterogeneous fluorescence decay observed for the 6MI-labeled oligonucleotides. DAS revealed that the longer wavelength spectral shifts observed for G1 with quadruplex folding are associated with the longest fluorescence decay time &tau1, and appear to originate from enhanced solvent interactions around the G1 fluorophore. Environmental heterogeneity has been further examined using single value decomposition (SVD) analyses for the absorbance and fluorescence thermal folding profiles. We have extracted at least four components for the unlabeled HT4 sequence and at least three for the fluorescence melting profiles of the 6MI-labeled sequences. Our data suggest that the folding pathway for the quadruplex formation process involves intermediate states. Overall, our studies provide additional information and better understanding about the complex HT4 G-quadruplex conformational dynamics under physiological conditions. Such knowledge can assist in the designing of future anti-cancer drugs targeting the human telomeric quadruplex.
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.
The Role of the Striatal Neuropeptide Neurotensin in the Methamphetamine-induced Neural Injury in Mice
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Methamphetamine (METH) is a widely abused psychostimulant that induces neurotoxicity to several brain regions, including the striatum. Similar to dopamine (DA) in chemical structure, METH can be transported into DA pre-synaptic terminals, evoking the neurodegeneration in DA terminals and post-synaptic striatal neurons. Despite the critical role of DA in METH-induced neurodegeneration, no pharmaceutical therapeutics has been approved for these conditions. It is therefore essential to investigate the endogenous factors regulating the dopaminergic system. The neuropeptide neurotensin has emerged as a potential modulator of METH-induced striatal neurodegeneration mainly due to its intimate interactions with dopamine in the striatum. In this study, we investigated the role of the neuropeptide neurotensin on METH-induced striatal neurodegeneration in mice. We observed that a single injection of METH (30 mg/kg, ip) induced the loss of approximately 15% of striatal neurons. An agonist of the neurotensin receptor 1 (PD149163, ip) attenuated the METH-induced striatal neuron apoptosis in a dose-dependent manner, while exerted no effect on METH-induced dopamine terminal degeneration. Utilizing quantitative Real Time PCR, we showed that METH also up-regulated neurotensin gene expression by 96% in stratal neurotensin mRNA. These data demonstrate that neurotensin modulates METH-induced striatal apoptosis through neurotensin receptor 1 (NTR1) in the striatum. In addition, NTR1 agonist attenuated METH-induced hyperthermia and can also attenuated the striatal apoptosis independent of body temperature regulation. To further investigate the corresponding mechanisms, we assessed its effect on glial cell activation, nitric oxide accumulation and DARPP32 phosphorylation in the striatum, which are all believed to aggravate METH-induced neurodegeneration. We observed that the NTR1 agonist attenuated the effects of METH on each of these three bio-markers. Our results also show that the NTR1 agonist alone caused decrease in phosphorylation of DARPP32 at Thr34, while NTR1 antagonist (SR48692) per se increased the phosphorylation of DARPP32 at Thr34. Since the DARPP32 phosphorylation pathway is responsible for interpreting signals to striatal projection neurons, neurotensin possibly modulates DARPP32 phosphorylation through regulation of DA and glutamate neurotransmission. Finally, the agonist of neurotensin, PD149163, may be considered as a potential therapeutic for treatment of METH-induced neurotoxicity. (Supported by R01 DA020142 from NIH)
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.
DESIGN AND OPTIMIZATION OF A DE NOVO PROTEIN CHARGE SEPARATION DYAD
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The ever-increasing demand for cheap, plentiful energy to fuel the needs of a growing population requires research into alternative clean energy. Solar irradiation has the potential to power the planet many times over; the challenge is efficient capture and conversion of this energy source. Nature has already solved this problem with photosynthesis, which harvests solar irradiation converting it to stored chemical energy and is the source of the energy for life. The goal of my dissertation is to use de novo designed protein to mimic the charge separation system in photosynthesis. A stable protein scaffold will be designed and used to position photoactive cofactors at predetermined distances to yield a high efficient charge separation domain. The creation of a simple single chain four helix bundle protein capable of binding two to three distinct cofactors for use as a light-activated charge separation domain is described. This protein was de novo designed using biologically derived binary patterning with metal ligand coordination and cysteine modification to control cofactor placement. The use of a heme and zinc phthalocyanine cofactors allow for simple bis-histidine and mono-histidine binding sites as the differentiating factor positioning and the quinone is positioned using cysteine mutations. The domain has been expressed and combined with cofactors and biophysically characterized and preliminary data on electron transfer have been obtained.
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.