The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.

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<p>During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.</p><p>From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.</p> / Dissertation

Master of Science / Department of Kinesiology / Brett J. Wong / Nitrate supplementation in the form of beetroot juice (BRJ) has been shown to increase nitric oxide (NO), where nitrate can be reduced to nitrite and NO through both nitric oxide synthase (NOS) independent and dependent pathways. We tested the hypothesis that BRJ would augment the NO component of cutaneous thermal hyperemia. Dietary intervention consisted of one shot of BRJ for three days. Six subjects were equipped with two microdialysis fibers on the ventral forearm and randomly assigned to lactated Ringer’s (control) or continuous infusion of 20mM L-NAME (NOS inhibitor). The control site was subsequently perfused with L-NAME once a plateau in the local heating response was achieved to quantify NOS-dependent cutaneous vasodilation. Skin blood flow via laser-Doppler flowmetry (LDF) and mean arterial pressure (MAP) were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to %CVCmax. Maximal vasodilation was achieved via local heating to 43°C and 54mM sodium nitroprusside infusion. There was a significant decrease in DBP after BRJ (Pre-BRJ:74 ± 1 mmHg vs. Post-BRJ: 61 ± 2 mmHg; p < 0.05) and significant reduction in MAP after BRJ (Pre-BRJ: 90 ± 1 mmHg vs. Post-BRJ: 80 ± 2 mmHg; p < 0.05). The initial peak and secondary plateau phase of cutaneous thermal hyperemia were attenuated at sites with continuous LNAME; however, there was no effect of BRJ on either the initial peak at control sites (Pre-BRJ: 76 ± 3%CVCmax vs. Post-BRJ: 75 ± 4%CVCmax) or L-NAME sites (Pre-BRJ: 60 ± 4%CVCmax vs. Post-BRJ: 59 ± 5%CVCmax) or the secondary plateau phaseat control sites (Pre-BRJ: 88 ± 4%CVCmax vs. Post-BRJ: 90 ± 4%CVCmax) or L-NAME sites (Pre-BRJ: 45 ± 5%CVCmax vs. Post-BRJ: 51 ± 3%CVCmax). The decrease in %CVCmax to L-NAME infusion during the plateau of local heating (i.e. post-L-NAME drop) was greater after BRJ (Pre-BRJ: 36 ± 2%CVCmax vs. Post-BRJ: 28 ± 1%CVCmax; p < 0.05). This resulted in a greater contribution of NOS to the plateau phase of local heating (Pre-BRJ: 57±3%CVCmax vs. Post-BRJ: 64±2%CVCmax; p < 0.05). These data suggest BRJ modestly improves NOS-dependent vasodilation to local heating in the cutaneous vasculature of healthy humans.

Glioblastoma is the most malignant form of brain cancer. Due to its aggressive nature, extensive research has been performed, but little progress has been made in identifying effective treatment options. Glycogen synthase kinase-3 (GSK-3) is a ubiquitous, multifaceted protein kinase. Previous studies have shown that small molecule inhibitors of GSK-3 block the migration of glioblastoma cells and may prevent spread of tumor in the brain. However, these studies were performed using non-selective GSK-3 inhibitors (LiCl and an indirubin derivative, BIO); thus, it was unclear whether GSK-3 was the most important target. In this study, we used recently generated highly selective GSK-3 inhibitors (CHIR99021, AZD1080, and AZD2858, as well as BIO) to investigate these questions. These were applied to four glioblastoma cell lines: G30, G9, U251, and U1242, in three migration assays: transwell, spheroid, and wound healing (scratch) assay to further assess the suitability of GSK-3 as a target in glioblastoma. We also utilized the ATP Luciferase reporter assay for cell viability to assess the influence of our panel of drugs on cell migration versus viability. In addition, the TOPFlash Luciferase reporter assay was performed as an indicator of the level of GSK-3 inhibition. The TOPFlash assay showed that all GSK-3 inhibitors were able to increase luciferase levels. This indicates that GSK-3 was inhibited in our cells after drug treatment. The transwell assays showed us that the GSK-3 inhibitors were able to block migration significantly in all cell lines tested in a dose-dependent manner. The effectiveness of GSK-3 inhibition in the three-dimensional collagen spheroid assays was cell line-dependent, with the non-selective GSK-3 inhibitor BIO showing the most potent effects. Cell migration was not blocked by any of the three selective GSK-3 inhibitors in the wound healing scratch assay. Thus we have found that the three distinct highly selective inhibitors of GSK-3 block glioblastoma cell migration, but only work consistently in the transwell assay. Therefore, we conclude that GSK-3 might be important in the contraction and morphological changes necessary for glioblastoma cells to migrate through the 8 micron pores in the transwell. Further investigation into this observation is necessary. Though results were variable between assays, we conclude that the inhibition of GSK-3 is a promising potential therapeutic strategy for glioblastoma treatment.

Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L-citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)-5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.

RNA contains over 100 post-transcriptional chemical modifications. Of these, the most abundant is pseudouridine (Ψ), the 5’ ribosyl isomer of uridine. The formation of Ψ occurs at the polynucleotide level, and is catalyzed by Ψ synthase enzymes. To date, five families of Ψ synthases have been identified. Our work deals with the fifth and last family to be discovered, the Ψ synthase TruD family. TruD homologs are present in organisms across the three kingdoms of life. A sequence alignment of TruD homologs reveals distinct sequence insertions at specific and conserved locations in homologs from higher organisms. We have carried out extensive bioinformatics searches in an effort to characterize these insertion sequences, and have found that one of these insertions has a high degree of similarity to the R3H single-strand nucleic acid binding domain. To further understand the nature of this insertion, we examined its role in the enzymatic activity of the yeast TruD homolog, Pus7p, and found that mutating this insert decreased the enzyme’s activity by almost half. The human genome codes for two TruD homologs, PUS7 and PUS7L. These putative pseudouridine synthases were named based on their similarity to the yeast TruD homolog Pus7p, but their function has only been inferred based on sequence, and neither their activity, nor their structure have been examined. In an effort to further our understanding about the TruD synthase family, we have taken a closer look at PUS7 and PUS7L. We have determined the optimal conditions for over-expression of the enzymes in a bacterial expression system, and shown that the proteins are soluble, and can be purified using serial chromatography. In addition, we have queried these enzymes’ ability to recognize canonical TruD substrates and found that they are unable to complement an E. coli truD deletion or an S. cerevisiae pus7 deletion. Instead, the human PUS7 enzyme proved to be highly specific to the human tRNAGlu sequence, displaying activity targeted to U13. This activity appears to be independent of accessory factors. Taken together, this work strives to further our knowledge of Ψ synthases by examining TruD homologs present in higher organisms. TruD has the least sequence identity to the other four synthase families and does not possess any of the known RNA binding motifs. This work expands our knowledge base of the TruD family, the most divergent of the five Ψ synthase families.