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STRUCTURAL SEPARATIONS OF ENDOGENOUS AND EXOGENOUS COMPOUNDS DIRECTLY FROM TISSUE SECTIONS BY ION MOBILITY MASS SPECTROMETRYParson, Whitney Beth 04 August 2010 (has links)
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can provide the spatial distribution, relative abundance, and molecular identity of thousands of analytes directly from tissue sections. Due to the complex nature of tissue samples, additional analyte separation is required to increase the specificity of MALDI MS for analytes of interest. Here, the addition of a post-ionization gas-phase structural separation performed by ion mobility (IM) prior to MS is evaluated for its utility to enhance MALDI MS direct tissue analysis by (1) separating analytes of interest from other endogenous compounds, (2) simultaneously analyzing phospholipids and peptides, (3) performing simultaneous fragmentation of all ions, and (4) obtaining analyte gas-phase structural information. Current applications of MALDI IM-MS for tissue analysis as well as future research directions are provided.
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STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE U-BOX DOMAIN OF THE E4B UBIQUITIN LIGASENordquist, Kyle Andrew 31 August 2011 (has links)
Ubiquitination is a post-translational modification, that functions in a variety of cellular signaling pathways. Unlike modification with a small functional group by a single enzyme, modification by the small protein ubiquitin requires a three-step enzymatic cascade involving E1 activating, E2 conjugating, and E3 ligating enzymes. While much information is known about this process, a comprehensive understanding of the molecular mechanisms of ubiquitination is lacking. This thesis investigated the structure and the function of the U-box E3 ligase, E4B, focusing on the interaction with the E2 enzyme and the activation of the E2~Ub conjugate by the E3 ligase.
In order to study E4B interaction with the E2 conjugating enzyme UbcH5c, I determined the three-dimensional solution structure of the U-box domain of E4B (E4BU) by NMR spectroscopy. Then, NMR chemical shift perturbation analysis was used to map the interaction with UbcH5c. This data, along with the new E4BU structure and the known structure of UbcH5c, was used to generate a computational model of the E4BU-UbcH5c complex. The oligomerization state of E4BU was also investigated; unlike other U-box E3 ligases, it was found that E4BU is monomeric. To determine if E4BU was functional as a monomeric E3 ligase, an in vitro autoubiquitination assay with UbcH5c was developed. These experiments showed that E4BU does function as a monomer.
The favorable characteristics of E4BU (small size, soluble, excellent NMR spectrum) make it an excellent model system to study the mechanism of E3 activation of the E2~Ub conjugate. Consequently, NMR chemical shift perturbation analysis was applied to the E4BU-UbcH5c~Ub complex. These results suggested the E2~Ub conjugate is activated by the E3 through an allosteric network. This model was tested by mutations made within this network, which significantly inhibited ubiquitination function. Initial studies of the dynamics of the UbcH5c~Ub conjugate in the absence and presence of E4BU were also undertaken, setting the stage for more detailed understanding of the role of dynamics in E3 activation of the E2~Ub conjugate.
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MECHANISTIC DETAILS OF THE PH-DEPENDENT ASSOCIATION OF BOTULINUM NEUROTOXIN WITH MEMBRANESMushrush, Darren 06 September 2011 (has links)
Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pH-dependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we have investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C-terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. Electron paramagnetic resonance analyses of spin labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH.
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Assessing the Components of the eIF3 Complex and Their Phosphorylation StatusFarley, Adam Richard 09 December 2011 (has links)
BIOCHEMISTRY
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ASSESSING THE COMPONENTS OF THE eIF3 COMPLEX AND THEIR PHOSPHORYLATION STATUS
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ADAM RICHARD FARLEY
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Dissertation under the direction of Professor Andrew J. Link
<p> The eukaryotic initiation factor 3 (eIF3) is a highly conserved multi-protein complex that is an essential component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, I examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. I hypothesized that for eIF3, there are unexpected and unidentified eIF3 protein-protein interactions and protein phosphorylations that regulate its function and activity in the process of translation initiation. 2-D LC-MS/MS mass spectrometry analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) co-purify with the core complex. These novel identifications expand the knowledge base for what is known about the function of eIF3 in yeast and expands its role to additional steps in protein synthesis.
<p> In vivo metabolic labeling of proteins with 32P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, I identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide at T191. Additionally, I used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. I found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, I showed that CK2 phosphorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype. Quantitative growth assay studies revealed that mutant strains lacking the phosphorylation have a doubling time that is increased by 33% relative to a control yeast strain. I propose that the observed phosphorylations stabilize interactions with the Prt1 protein as this region of Nip1 is suspected to be involved in Nip1-Prt1 contacts.
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BIOCHEMICAL AND STRUCTURAL ANALYSIS OF SV40 LARGE T ANTIGEN: INSIGHTS INTO CHAPERONE MEDIATED INACTIVATION OF RETINOBLASTOMA TUMOR SUPPRESSOR PROTEINWilliams, Christina Kay 28 March 2012 (has links)
Simian Virus 40 uses its Large T antigen protein to bind and inactivate retinoblastoma tumor suppressor proteins, yielding cell transformation. T antigen is a modular protein with four domains connected by linkers. The N-terminal domain, the J domain, classifies T antigen as a chaperone protein and is necessary for the inactivation of Rb. Physical interaction with Rb is mediated primarily by an LXCXE consensus motif immediately C-terminal to the J domain. An approach integrating nuclear magnetic resonance (NMR) spectroscopy and small angle x-ray scattering (SAXS) was used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N260) that extends from the J domain to the adjacent origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the two domains in N260. Rb bound to a construct containing the LXCXE motif and the J domain confirmed that weak interactions exist between Rb and the J domain. Analysis of the interaction of Rb with N260 indicated that the OBD is not involved in binding Rb and that the J domain retains dynamic independence from the remainder of T antigen. These results support a T antigen chaperone model in which the J domain orientation changes as it acts upon protein complexes to promote cell transformation.
The ubiquitin ligase Cullin 7 (Cul7) has been implicated in chaperone-mediated inactivation of Rb. It is known that Cul7 binds to T antigen via residues within the J domain. However, the Cul7 domain involved in this interaction is unknown. Yeast two hybrid screens in the Ellen Fanning laboratory suggest that the CPH domain of Cul7 bound to T antigen. NMR chemical shift perturbation experiments confirmed the interaction between the J domain of Tag and CPH domain of Cul7. These data identified specific CPH domain residues involved in binding to the Tag J domain, which map to a specific binding surface. Our results set the stage for mutational analysis to establish the functional role of the Tag-Cul7 interaction.
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Identification and Characterization of p53 Target GenesEby, Kathryn Grace 16 June 2010 (has links)
After 30 years of research, p53 is recognized as one of the most frequently mutated genes in human cancer (Baker et al, 1989; Nigro et al, 1989; Momand et al, 2000; Daujat et la, 2001). To accomplish its tumor suppressive role, p53 transcriptionally regulates a multitude of genes involved in cell cycle progression, genomic stability, cellular senescence, apoptosis, angiogenesis, cell migration, and autophagy. To date approximately 150 genes are documented as direct p53 target genes. The goal of this dissertation research was to identify novel p53 family transcriptional targets and determine their functions in biologically-relevant processes downstream of the p53 family signaling axis.
In Chapter III of this dissertation, I describe the use of statistical and bioinformatic tools to perform genomic analyses and identify a subset of novel putative p53 transcriptional targets. With the overlay of genomic datasets and predictive mathematical models, I developed a panel of high-confidence p53 transcriptional targets. I describe current and future plans to analyze these target genes, in a high-throughput manner, and identify the contribution of each target gene to p53-regulated processes such as cell cycle arrest, apoptosis, and autophagy, among others.
In Chapter IV of this dissertation, I describe the identification of ISG20L1 as a target gene of p53 as well as family members p63 and p73. Ectopic expression of ISG20L1 decreased clonogenic survival, but changes in ISG20L1 protein levels did not alter apoptosis. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical and immunohistochemical measurements of LC3-II.
Investigation of genes and signaling pathways involved in cell death associated with autophagy is critical given the keen interest in targeting autophagy as an anticancer therapeutic approach in tumor cells that are defective in apoptosis.
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Identifying and defining the genome maintenance functions of SMARCAL1Robbins, Carol Bansbach 15 June 2012 (has links)
In this dissertation I identify and define SWI/SNF, matrix-associated, actin-dependent regulator of chromatin, A-like 1 (SMARCAL1) as a genome maintenance protein. First, I introduce a functional genomic screen designed to identify novel genome maintenance genes. This screen led to the identification of SMARCAL1 and is the foundation for my project, providing the preliminary evidence to support that SMARCAL1 functions to maintain genome integrity. Next, I investigate the specific genome maintenance activity of SMARCAL1 and find that localization of its enzymatic activity to stalled replication forks is required for genome integrity during DNA replication. Finally, I explore how phosphorylation of SMARCAL1 regulates its function. Importantly, mutations in SMARCAL1 cause the human disease Schmike immuno-osseous dysplasia (SIOD). The research presented herein suggests that the phenotypes of SIOD are, in part, due to defects in SMARCAL1 genome maintenance activity.
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Estrogen receptor activation factors (E-RAF) of the goat and rat uteri: Structural and functional studiesKumar, Prem M 08 1900 (has links)
Estrogen receptor activation factors
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Estrogen receptors of the goat uterus: Emphasis on a receptor form that enters the nucleus as a constituent of heterodimerKarthikeyan, Narayanan 10 1900 (has links)
Emphasis on a receptor form that enters the nucleus
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Theoretical studies on protein sequencesRani, Meeta 12 1900 (has links)
Theoretical studies on protein sequences
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