Cysteinyl peptides likely played an important role in the prebiotic synthesis of cofactors, such as iron-sulfur clusters. However, cysteinyl peptides must be reduced in order to coordinate iron-sulfur clusters. Mixtures of ferric ions and cysteinyl peptides leads to the reduction of ferric to ferrous ions and the concomitant formation of disulfide bridged, oxidized cysteinyl peptides that are incapable of coordinating an iron-sulfur cluster. Here, we develop a photochemically driven solution to this problem. Lipoic acid (( R )-5-(1,2-dithiolane-3-yl)pentanoic acid), a molecule structurally similar to fatty acids, can be photochemically reduced and can subsequently reduce the oxidized cysteinyl peptides needed for the coordination of an iron-sulfur cluster. Other dithilane ring containing molecules possess similar activity to lipoic acid. The synthesis of small peptides containing cysteines, such as glutathione and GCG (Gly-Cys-Gly) is easy by both solid phase and solution phase methodologies. However, as the length of the peptide increases, the yield begins to decrease, especially for peptides containing cysteines due to oxidation. One solution could be to exploit a previously uncovered mechanism for the joining of peptides into longer peptides. Such mechanisms, referred to as CPL for catalytic peptide ligation, rely on either thiols or metals as catalysts and peptide nitriles as substrates. Thus far, CPL has only been exploited with non-cysteinyl peptides. In this thesis, we extend CPL to cysteine containing peptides by taking advantage of the templating effects of Zn2+. Longer peptides with properly spaced cysteines are frequently better able to stabilize iron-sulfur clusters in aqueous solution than shorter peptides. Coordination can either be complete or an open coordination position, filled by solvent, can be used to bind substrate. Two well-known examples of such an arrangement are the radical SAM (S-adenosylmethionine) enzyme and aconitase being an enzyme of the citric acid cycle. We designed and synthesized peptide sequences that could coordinate a [4Fe-4S]2+ cluster with three cysteinyl ligands, leaving an open coordination position. The stability of the [4Fe-4S] cluster was affected by the intermediates of the citric acid cycle. The iron-sulfur can be reconstituted with the long peptidyl sequences from proteins such as SLC25A39 which contains four cysteine ligands to form [2Fe-2S] cluster, which is necessary for glutathione transport from cytosol to mitochondria.

The work in this thesis mainly focuses on the assessment of density functional methods for computing structures and energies of organic and bioorganic molecules. Previous studies found dramatic conformational and stability changes from B3LYP to MP2 geometry optimization for some Tyr-Gly conformers. Possible reasons could be large intramolecular basis set superposition errors (BSSEs) in the MP2 calculations and the lack of dispersion in the B3LYP calculations. The fragmentation method and three kinds of rotation methods were used to investigate intramolecular BSSE. It is concluded that the rotation method cannot be used to correct intramolecular BSSE along a rotation profile. Another methodology is to employ modern density functionals. We focused on M06-L with the Tyr-Gly conformer ‘book6’. Potential energy profiles were determined by computing the energy for geometries optimized at various fixed values of a distance that controls the degree of foldedness of the structure. M06-L manifested itself as a very promising method to investigate the potential energy surface of small peptides containing aromatic residues. To predict Tyr-Gly structures, 108 potential conformers were created with a Fortran program. The geometry optimizations were done using M06-L/6-31G(d) and M05-2X/6-31+G(d). Two schemes were employed and the most stable conformers were compared to the 20 stable conformers found by B3LYP. Both schemes found 10 conformers similar to one of the B3LYP stable conformers, as well as several newly found conformers. The study of a missing B3LYP stable conformer showed that the possible reason of missing conformers may be the lack in dispersion in B3LYP theory. To study the hydration effect, we studied the conformations of neutral and zwitterionic 3-fluoro-γ-aminobutyric acid (3F-GABA) in solution using different solvation models, mainly the explicit water molecule models. Zwitterionic forms of 3F-GABA are preferred in solution. M06-2X performs better in calculating transition energy profiles than MP2.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 140-150).

No description available.

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation. We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation. / Graduation date: 2012

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 137-167).

Long-time research of chronologically aging yeast populations of Saccharomyces cerevisiae laboratory strains revealed that yeasts are able to differentiate into specialized cell types. Differentiation of liquid cultures growing in glucose rich medium and differentiation of colonies growing on solid glycerol medium has been previously studied. These populations create two fractions of cells with diverse morphology which adapt their metabolism and physiology to enable a long-term survival of the yeast population in environment with limited nutritional potential. In this study, yeast subpopulations isolated from colonies growing on solid glucose medium and liquid cultures cultivated in glycerol medium were characterized. Newly isolated cell types were compared with already known cell types isolated from colonies and liquid cultures. Selected metabolic processes and stress resistance were analysed in studied populations. Based on previous studies of yeast differentiation, a spectrum of GFP-labelled marker proteins was choosen and production and localization of these marker proteins was monitored within yeast populations. Results of the analyses showed that type of medium and cultivation influence development and metabolism of each yeast cell type. Key words: Saccharomyces cerevisiae, BY4742, cell...

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.