Thesis (Ph.D.)--Ohio University, November, 2006. / Includes bibliographical references (leaves 155-168).

Techn. Hochsch., Diss., 2003--Aachen.

Universiẗat, Diss., 2003--Freiburg (Breisgau).

Although F1-ATPase is extremely conserved among organisms, a putative subunit p18 was identified in Trypanosoma brucei F1-ATPase complex. To explore its function in the procylic, bloodstream and dyskinetoplastic trypanosomes, three different RNAi cell lines were created. Upon p18 silencing the F1-moiety structural integrity was impaired suggesting that p18 is indeed a bona fide subunit of this complex. Since F1-ATPase is crucial for the bloodstream form survival, its potential inhibitor from the 4-oxopiperidine-3,5-dicarboxylates class (JK-11) was examined. JK-11 inhibited growth of the bloodstream trypanosomes, decreased mitochondrial membrane potential and reduced ATPase and ATP synthase activity in mitochondrial lysates. Our results suggest that JK-11 may act on FoF1-ATP synthase/ATPase and its inhibition may contribute to the cytotoxicity of this drug.

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the TψC stem. In the halophilic euryarchaeon Haloferax volcanii, tRNATrp and tRNAMet, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and ψ54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNATrp Cm34 formation being guided by its own intron, while that of tRNAMet being guided by a unique guide RNA called sR-tMet. We created genomic deletion of H. volcanii tRNATrp intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. ψ54, the near universal modification found in TψC stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, Methanocaldococcus jannaschii (MjPus10) and Pyrococcus furiosus (PfuPus10) have differential activities towards ψ54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA ψ54 formation. Finally, as an extension to this study, we have identified a novel tRNA ψ54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10.

Methylgloxal synthase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been found in several Enterobacteriaceae. The enzyme along with glyoxalase I and II and D-lactate oxidase, therefore, constitute a nonphosphorylated shunt of the normal glycolytic pathway

Après un repas, la glycolyse, la lipogenèse et particulièrement 2 enzymes sont sollicitées : la GlucoKinase (GK) et la Fatty Acid Synthase (FAS) augmentant la biosynthèse des acides gras (AG). Une autre voie du glucose conduit à la O-GlcNAc, glycosylation assurée par l’OGT et l’OGA. Au vu de la relation entre le glucose, la O-GlcNAc et le métabolisme glucido-lipidique, la O-GlcNAc contrôle probablement l'expression et l'activité de la GK et de la FAS. L’objectif a été de caractériser la O-GlcNAc des 2 enzymes et l’impact de la modification sur leurs propriétés. Les niveaux de O-GlcNAc, de GK et de FAS ont été mesurés dans différents modèles : des foies et des hépatocytes primaires de souris et des lignées cellulaires hépatiques cultivés dans des conditions de O-GlcNAc variables. Nous démontrons que la FAS et la GK sont O-GlcNAc en fonction des conditions nutritionnelles, en corrélation avec une meilleure stabilité de la protéine. Un lien existerait entre l’apport excessif de glucose, l’augmentation de O-GlcNAc et la production d’AG conduisant à la stéatose. Les perspectives sont l’étude de la régulation de la FAS par O-GlcNAc au cours du cycle cellulaire. La FAS est primordiale dans la biosynthèse d’AG, composants majeurs des membranes plasmiques. Par conséquent, elle est dérégulée en cas de prolifération cellulaire anormale, caractéristique des cellules cancéreuses. Ce défaut de prolifération est accompagné d’une augmentation de la FAS, de l’OGT et des protéines O-GlcNAc. Nos premiers résultats montrent que l’expression de la FAS varie au cours du cycle. L’OGT y joue probablement un rôle puisque son inhibition dérégule les variations de la FAS au cours du cycle cellulaire. / After a meal, the glycolysis, the lipogenesis and particularly two enzymes are activated: Glucokinase (GK) and Fatty Acid Synthase (FAS) causing an increase in fatty acid biosynthesis. Another pathway of glucose leads to O-GlcNAc, glycosylation catalysed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In view of the relationship between glucose levels, O-GlcNAc and the glucido-lipidic metabolism, the O-GlcNAc would regulate the expression and activity of GK and FAS. The aim was to characterize O-GlcNAc of GK and FAS, and the impact of this modification on their properties. O-GlcNAc, GK and FAS levels were measured in various models: livers and primary hepatocytes of mouse and liver cell lines cultured in conditions that modulate O- GlcNAc levels.We demonstrated that FAS and GK are O-GlcNAc depending on nutritional conditions in correlation with a better stability of proteins. It must exist a link between an excessive intake of glucose, increased levels of O-GlcNAc and abundant fatty acid production leading to hepatic steatosis. In the perspectives, we focused on the regulation of FAS expression by O-GlcNAc during the cell cycle. Indeed, FAS plays a pivotal role in the biosynthesis of biological membranes fatty acids. Accordingly, FAS may be dysregulated in abnormal cell proliferation, a major characteristic of cancer cells. In addition, these cells exhibit an overall increase of OGT expression and O-GlcNAc protein. Our initial results suggest that FAS has a variable expression during cell cycle. In addition, OGT and O-GlcNAc may play a role since the use of an OGT inhibitor deregulate changes in the FAS expression in different phases of the cycle.

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Ergothioneine, a histidine-derived thiol, protects cells against reactive oxygen species and is emerging as a longevity vitamin. Ovothiol, another histidine-derived thiol, is also a potent antioxidant with therapeutic potential due to its anti-inflammatory and anti-proliferative activities. Despite these promising health benefits, the production of ergothioneine is limited by the underlying challenges of its only industrial synthetic method, while ovothiol is not commercially available. Due to these issues, the production of these thiols through metabolic engineering/synthetic biology approaches is appealing. The central steps in the ergothioneine and ovothiol biosynthetic pathways are the oxidative coupling C-S bond formation reaction mediated by non-heme iron sulfoxide synthases, and the pyridoxal-5'-phosphate (PLP)-dependent C-S lyases. This sulfur transfer strategy differs from all other pathways reported. Therefore, these trans-sulfuration reactions in ergothioneine and ovothiol biosyntheses are significant from both basic and translational research perspectives, hence, they were selected as my thesis project. This thesis comprises of five chapters. Sulfur metabolism and the biosynthesis of sulfur-containing natural products are presented in Chapter 1. The computational-guided protein engineering of a thermophilic sulfoxide synthase (EgtB) from Chloracidobacterium thermophiluim is covered in Chapter 2. Chapter 3 describes the mechanistic studies of the reductive C-S lyase (Egt2 from the Neurospora crassa’s ergothioneine biosynthesis), which revealed the involvement of a sulfenic acid intermediate in this reaction. In addition to reconstituting the ergothioneine biosynthetic pathway in vitro presented in Chapter 3, I fully reconstituted the in vitro ovothiol A biosynthetic pathway from Erwinia tasmaniensis, which is described in Chapter 4. In Chapter 5, the mechanistic studies of the ovothiol sulfoxide synthase OvoA using unnatural amino acid incorporation via amber-codon suppression are discussed. The success of this thesis work paves the way for the industrial production of ergothioneine and ovothiol through metabolic engineering/synthetic biology approaches. This study has also laid the foundation for future in-depth mechanistic characterization of these novel enzymes.

Studies were conducted in 1996 through 1999 to: (1) evaluate the responses of one imidazolinone (IMI)-susceptible (S) and four -resistant (R1, R2, R3, and R4) smooth pigweed populations to various acetolactate synthase (ALS)-inhibiting herbicides, (2) determine the mechanism of resistance, and (3) evaluate the relative growth and competitiveness of each population. Field studies were conducted in 1996 near Marion, MD, in a field with a history of repeated imazaquin use. Smooth pigweed control with IMI herbicides was < 8 percent, but control with sulfonylurea (SU) herbicides ranged from 73 to 99 percent. Follow-up greenhouse studies were used to confirm IMI resistance in the Marion, MD smooth pigweed population (R4) as well as three others (R1, R2, and R3). R populations were 730- to 1350-fold more tolerant to imazethapyr than the S population. Based on resistance ratios, all R populations displayed low-level cross-resistance to chlorimuron and negative cross-resistance to thifensulfuron, pyrithiobac, and cloransulam-methyl with R2 being the most sensitive of the R populations to pyrithiobac and cloransulam-methyl. Absorption, translocation, and metabolism of ¹⁴C-cloransulam-methyl in S and R2 populations were generally similar. Three metabolites of cloransulam-methyl with ratio of front (Rf) values approximately 0.83, 0.65, and 0.45 were isolated. The metabolite with a 0.83 Rf value increased over time as the parent molecule decreased indicating that it plays a major role in cloransulam-methyl metabolism in smooth pigweed. The other metabolites did not change significantly over time and never represented more than 5 percent of the extracted radioactivity. The identity of these metabolites has not been determined. Using enzyme assays, it was determined that IMI resistance in R populations was due to an altered ALS that was no longer susceptible to inhibition by these herbicides. ALS from S, R1, and R2 populations responded similarly to chlorimuron and thifensulfuron, but reductions in enzyme activity by chlorimuron and thifensulfuron were significantly greater for R3 ALS than for S, R1 or R2 ALS. ALS from R2 and R3 was significantly more sensitive to inhibition by pyrithiobac compared to S ALS. Based on resistance ratios, R2 and R3 ALS were also more sensitive to inhibition by cloransulam-methyl than S ALS. Negative cross-resistance to thifensulfuron, pyrithiobac, and cloransulam-methyl in some R populations at the whole-plant level can be explained by increased sensitivity at the enzyme level. Under noncompetitive conditions in the greenhouse, S produced 17, 23, 25, and 44 percent more biomass than R1, R2, R3, and R4 populations, respectively. S plants were also taller than R plants 17 and 21 d after planting (DAP) and displayed a faster initial rate of leaf area increase compared to all R populations. The net assimilation rate of S was significantly higher than R2 and R3 populations 24 DAP. R3 and R4 populations had significantly less chlorophyll per g of plant tissue compared to S; therefore, reduced growth in some R populations compared to S may be linked to chlorosis that generally appears early in seedling development. Biomass production in the field under competitive conditions was similar for all populations using both monoculture and mixed populations. For this reason, the differences in growth observed in the greenhouse in the S population may not confer a competitive advantage over R populations in the field. / Ph. D.