Browsing by Subject "APOBEC3G"

Now showing 1 - 5 of 5
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    Brightness analysis in finite geometries: probing protein interactions in cellular, cell-free and aqueous environments
    (2012-12) Macdonald, Patrick
    Fluorescence fluctuation spectroscopy (FFS) is a powerful technique for quantitatively analyzing protein interactions. Using brightness analysis methods, we are uniquely able to measure the stoichiometry of protein complexes. FFS is particularly valuable because it allows measurements within living cells. This thesis demonstrates that measuring in very small volumes, such as E. coli cells, introduces a bias into the measured brightness. We show that this bias is a result of accumulative sample loss, or photodepletion, and that we can account for this effect and recover correct brightness values. Similarly, very thin samples, such as cell cytoplasm, introduce a bias due to the sample being shorter along the vertical axis than the volume of the excitation light. We introduce z-scan FFS and theory to identify and model thin samples and to recover unbiased data. Although measuring in cells is a primary strength of the FFS technique, some studies require the greater degree of experimental control afforded by solution measurements. Thus, we characterize cell-free expression solution for FFS measurements, an environment that offers increased control but permits genetic fluorescent labeling. We take advantage of this system to perform chromophore maturation experiments as a function of temperature on three common fluorescent proteins: EGFP, EYFP and mCherry. Our results prove that EGFP has fast maturation and is a good reporter for fluorescence experiments. Finally, we apply FFS and brightness analysis to the enzyme, APOBEC3G. We reveal that APOBEC3G interactions with RNA and single-stranded DNA are sequence dependent, which has important implications for the mechanism by which APOBEC3G packages itself into HIV-1 viral particles and restricts the virus to prevent infection.
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    High-throughput Screening and Chemical Synthesis for the Discovery of APOBEC3 DNA Cytosine Deaminase Inhibitors
    (2015-09) Olson, Margaret
    APOBEC3 (APOBEC3A-APOBEC3H) enzymes catalyze single-stranded (ss)DNA cytosine-to-uracil (C-to-U) deamination as a function of innate immune defense against foreign DNA. Host cellular protection results from APOBEC3-catalyzed lethal mutagenesis of the offending genome. When misregulated, however, APOBEC3 enzymes have been demonstrated to drive the genetic evolution of numerous cancers and HIV-1. Specifically, sub-lethal levels of APOBEC3D/F/G/H-catalyzed mutation can enable HIV-1 escape from immune defense and antiretroviral therapies. Moreover, APOBEC3B over-expression in breast, bladder, cervical, lung, and head/neck cancers generates high levels of C-to-U mutation, which drives tumor formation, metastasis, and chemotherapeutic resistance. Thus, small molecule inhibition of APOBEC3-catalyzed deamination may provide a novel strategy for HIV-1 and cancer drug development. This thesis highlights efforts to discover small molecule inhibitors of the APOBEC3s through high-throughput screening (HTS) and chemical synthesis. Chapter 2 discusses an HTS of 168,192 compounds against APOBEC3B and APOBEC3G. In this effort, MN23 was discovered as a potent inhibitor of APOBEC3B (IC50 = 150 nM) and APOBEC3G (IC50 = 5.5 µM). Chapter 2 also reports a novel synthesis of MN23, and preliminary efforts to elucidate its mechanism of inhibition. Chapter 3 presents a class of covalent APOBEC3G-specific inhibitors based on a 1,2,4-triazole-3-thiol substructure. This compound class is predicted to inhibit APOBEC3G by covalently binding C321, forcing an inhibitory conformational change within the enzyme active site. Chapter 4 reports the discovery of a novel APOBEC3G inhibitory chemotype, which was discovered from the deconvolution of an impure HTS hit. In this effort, we also identified a previously unreported Pan Assay Interference Scaffold (PAINS), and characterized the mechanism by which compounds of this class undergo oxidative decomposition. Finally, Chapter 5 describes how benzthiazolinone-based APOBEC3 inhibitors are being developed into probes of APOBEC3 structure and function. Brief descriptions of two unrelated projects performed concurrent with these studies are also detailed in the Appendices.
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    Human immunodeficiency virus evasion of APOBEC3 restriction factors
    (2012-10) Albin, John Squire
    The human immunodeficiency virus accessory protein Vif protects the viral genome from the mutational activity of APOBEC3 subfamily DNA cytosine deaminases by facilitating their proteasomal degradation, thereby preserving viral infectivity. A comprehensive understanding of the components of the Vif-APOBEC3 interaction is therefore important for consideration of the potential for novel antiretroviral approaches aimed at modulating this critical host-pathogen interaction. Here, we establish APOBEC3F among the seven subfamily members as a valid model for the study of the APOBEC3-Vif interaction. By utilizing this model as a starting point, we further define the APOBEC3-Vif interaction sites in each protein and the downstream ubiquitin acceptor sites modified en route to APOBEC3 degradation, in the process deriving broader insights into the nature of the interactions between different APOBEC3 proteins and Vif. In contrast with the diversiform APOBEC3-Vif interactions proposed in the extant literature, we find that the interaction of Vif with different APOBEC3 proteins likely proceeds through a conserved helix-helix interaction. Even if one were to successfully block this interaction for therapeutic purposes, however, the virus may develop accessory mechanisms of APOBEC3 evasion to bypass the intervention. While we find that this can occur, present evidence suggests that such alternatives may be insufficient to circumvent restriction in cells that naturally express multiple APOBEC3 proteins. Thus, it may be possible to potentiate the action of multiple endogenous antiretroviral proteins to counteract human immunodeficiency virus infection by targeting a conserved interaction motif as described herein.
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    Structural and Mechanistic Insight into APOBEC3G DNA Binding and Deamination
    (2015-07) Solomon, William
    Humans express the APOBEC3 family of proteins to defend against endogenous and exogenous DNA pathogens. APOBEC3 proteins display significant activity towards HIV-1 through incorporation into budding viral particle and interacting with HIV's RNA genome. In order to stably integrate into the human genome, HIV reverse transcribes the single stranded RNA genome into a double stranded DNA genome through a single stranded DNA intermediate. Once bound to the transient single stranded viral DNA intermediate, APOBEC3 proteins deaminate cytidines to uridines. Transcription over the resulting mutations results in G to A transversions in the coding sequence of the virus and non-functional gene products. APOBEC3 proteins are highly active on the single stranded DNA intermediate but lack catalytic activity on cytidines present in the viral RNA. APOBEC3G is the most potent of these innate viral mutagens and selectively targets tri-cytidine hotspot motifs in viral genome intermediates. When I began my thesis research, the effects of A3G mutagenesis had been extensively studied but the structural interactions with single stranded DNA and the mechanism of RNA exclusion were unknown. My thesis research helped identify the amino acid responsible for pH dependent effects on substrate binding. The identification of this residue allowed for the development of a pH insensitive variant of A3G that provided indirect evidence for a reduced pH in the viral capsid during the initiation of reverse transcription. These results, along with the kinetic characterization of A3Gctd and determination of the substrate factors crucial for deamination, are described in Chapter 2. In chapter 3, I continued to explore the effects of this pH dependent increase in binding affinity. I utilized NMR spectroscopy to identify the structural interactions of catalytic substrate binding. By generating a structurally stable but catalytically inactive mutant, I was able to identify differences between substrate interactions. This mutant also allowed me to explore structural interactions involved in substrate recognition and RNA exclusion. This lead to the identification of a novel substrate and a ribose sugar pucker dependent mechanism for target discrimination. The role of APOBEC3 proteins in retroviral restriction as well as their connection to several types of cancer makes them a prime target for therapeutic interventions. My thesis research in trying to understand the structural and mechanistic components of the APOBEC3/DNA interaction provides information that may be useful in the development of treatments targeting this family of proteins.
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    Structural studies of the deaminase domain of the human HIV-1 restriction protein APOBEC3G
    (2010-09) Chen, Kuan-Ming
    According to a report from the UNAIDS on the global AIDS epidemic in 2009, about 33.4 million people were living with the HIV virus, with a 2.7 million increase each year. HIV can lead to AIDS, which is a set of symptoms and infections resulting from the damage to the human immune system. However, due to the rapid mutability and productivity of the HIV, identifying treatments and therapeutic intervention remains challenging and has seen limited progress. In the past year, one novel innate defense against HIV infection was discovered, in which the human protein APOBEC3G (A3G) plays an important role. A3G was identified as a single-strand DNA deaminase that potently inhibited the replication of the HIV-1ΔVif virus. It produces the nonfunctional provirus by deaminating the cytosines to uracils on the minus-strand viral cDNA. Consequently, A3G can genetically inactivate HIV and recent studies have demonstrated that this activity is as potent as any current anti-retroviral drug. However, the exact model of this mechanism at atomic level has not yet been elucidated due to the low solubility of A3G which presents an obstacle for biochemical and structural studies. The research in this thesis took a structural approach to screen for a catalytically active and more soluble C-terminal deaminase of APOBEC3G (A3G-ctd) derivative, determine the NMR solution structure of this variant (A3G-2K3A) and characterize its interaction with single-strand DNA. Due to intrinsically low solubility of A3G-ctd, a strategy to design more soluble derivatives of the catalytic domain was performed prior to NMR structure determination. Two key methods: a solubility test and a E. coli-based mutation assay were used to test the solubility and catalytic activity of APOBEC3G variants. Deletion mutant analyses of APOBEC3G found the minimal catalytic region consisted of amino acids 198-384 (A3G198-384). Various alanine and lysine substitution variants based on this fragment were constructed and examined to screen for improved solubility and enhanced activity. One variant A3G198-384-2K3A (L234K-C243A-F310K-C321A-C356A) showed a significant improvement in both assays, and was purified as a monomer. The three-dimensional structure of A3G198-384-2K3A was then determined by triple resonance NMR spectroscopy. It consists of five β-strands that form a hydrophobic platform surrounded by five α-helices. Summarizing the DNA titration data, E. coli-based catalytic activity, conserved residues and computational modeling, the DNA binding mechanism of A3G was proposed in which a canyon formed by positively charged residues guides single-strand DNA binding and positions the target cytidine for deamination. Subsequently, a longer catalytic domain, A3G191-384-2K3A, was found to have higher activity than that of the A3G198-384-2K3A derivative. The longer domain has an additional α1-helix (residues 201-206) that was not observed in the shorter variant and part of the last α-helix (residues 191-194) of the N-terminal domain. The truncated model of the N-terminal domain was generated from the C-terminal NMR structures based on the sequence homology. Finally, a novel full-length A3G model was constructed by physically overlapping the α-helix (residues 191-194) of the N-terminal domain model and the C-terminal domain structure.