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Identification of a sequestered mitochondrial aconitase derived peptide recognized by virus-specific cytolytic T lymphocytes /Fan, Rong. January 1999 (has links)
Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Sequestered self peptide. Includes bibliographical references (p. 134-154). Also available online through Digital Dissertations.
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Functional analysis of a family of proteins implicated in Trypanosoma brucei lifecycle progressionDean, Samuel January 2008 (has links)
Bloodstream trypanosomes initiate differentiation to procyclic forms in response to a citrate/ cis-aconitate (CCA) signal. A cell line was previously selected (“defective in differentiation-clone 1”; DiD1) that was unable to differentiate to procyclic forms (Tasker et al. (2000)). Additionally, expression profiling of this line in comparison to the parental line by macroarray hybridisation identified two differentially-expressed transcripts from an 8 gene cluster of highly homologous genes we named PAD genes (Proteins Associated with Differentiation). Members of this family show distinct expression profiles throughout the trypanosome lifecycle at both the mRNA and protein level, and are localised to the cell surface membrane of the cell. At least 1 member of the family (PAD1) shows stumpy form specific RNA and protein expression, representing the first useful molecular marker for this stage, and exhibits biochemical specificity for citrate. Additionally, another member of this family (PAD2) is upregulated in response to low temperature, a condition reported to cause hypersensitivity to CCA. Finally, RNAi mediated ablation of the PAD gene transcripts compromised the capacity of stumpy form trypanosomes to differentiate to the procyclic form in response to CCA. These combined expression, cytological, reverse-genetic and biochemical data make PAD proteins excellent candidates for recognition of the signal to initiate differentiation in response to CCA.
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I. Understanding Membrane Interactions of Bacterial Exoproteins; II. Identification and Characterization of a Novel Mammalian cis-Aconitate DecarboxylaseCheng, Jiongjia January 2013 (has links)
Thesis advisor: Mary F. Roberts / Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-pi latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-pi box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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