The phosphothreonine lyase class of enzymes represents a recently discovered set of enzymes that catalyze a dephosphorylation reaction. The catalysis is carried out using a unique elimination mechanism without any involvement of cofactors. Crystallographic studies of SpvC, a phosphothreonine lyase, and its mutant show that the mutation of the general catalytic acid does not result in any significant perturbations to the tertiary and the secondary structure of the protein. Using results from the structural studies and a deuterium isotope exchange experiment, we conclude that the reaction catalyzed by SpvC may not involve formation of a carbanion at the active site.

ATP-citrate lyase (ACLY), upregulated in chronic kidney disease (CKD), catalyzes the synthesis of acetyl-coA from citrate. Acetyl-CoA is a vital precursor for lipid/cholesterol synthesis and histone acetylation that regulates gene expression. In renal cells, ACLY regulates fibrogenic, lipogenic and inflammatory gene expression; its inhibition reduced fibrosis in the unilateral ureteral obstruction (UUO) model. The ACLY metabolic by-product malonyl-coA is also an important inhibitor of fatty acid oxidation (FAO), and defective FAO in proximal tubular epithelial cells (PTEC) is now established as a major contributor to fibrosis. Here we tested the efficacy of a novel ACLY inhibitor on reducing fibrosis and its potential role in improving FAO in UUO. 8-week-old male C57BL/6J mice underwent UUO surgery and were treated orally with an ACLY inhibitor (EVT0185, Espervita Therapeutics) for 10 days. Kidneys were assessed by immunohistochemistry, immunoblotting, and RNAseq. Effects of ACLY inhibition were tested on the HK2 PTEC cell line and primary renal fibroblast responses to TGFβ1 (5ng/ml, 48h), a cytokine known to promote fibrosis and reduce FAO. Lipid accumulation was assessed by Oil Red O staining and LC/MS analysis. ACLY inhibition significantly and dose-dependently decreased fibrosis in the UUO model determined by trichrome, PSR, fibronectin, and α-smooth muscle actin (SMA) expression. ACLY inhibition decreased macrophage (F4/80) infiltration including that of the profibrotic M2 phenotype marked by CD206. RNAseq analysis showed upregulation of FAO-related hallmark pathways and reduction in inflammation pathways with ACLY inhibition. Defective FAO is known to result in PTEC apoptosis and lipid accumulation. ACLY inhibition reduced both apoptosis, as assessed by the presence of cleaved caspase 3, as well as lipid accumulation, with a particular decrease in cholesteryl esters. In HK2 cells and renal fibroblasts, TGFβ1-induced fibrotic protein expression was inhibited by ACLY inhibition, and lipid accumulation was reduced in PTECs. ACLY inhibition reduced renal fibrosis, apoptosis, and lipid accumulation in UUO mice. ACLY inhibition also prevented profibrotic responses to TGFβ1 in PTECs and fibroblasts. Current studies are ongoing to confirm beneficial effects on restoring FAO. / Thesis / Master of Science (MSc) / Chronic kidney disease (CKD) is the leading cause of kidney failure in Canada, affecting 4 million Canadians. There is no cure for CKD and current treatments are only able to slow down disease progression. CKD is caused by scarring in the kidney. The kidney requires a lot of energy to do its job filtering our blood and creating urine, and with CKD the ability to create and use energy is reduced. The protein ATP-citrate lyase (ACLY) that is present in the kidney contributes to CKD. Research has shown that people and mice with CKD have higher levels of this protein than healthy individuals. ACLY creates a molecule called acetyl-coA that is likely to cause our kidneys to produce less energy. This study will test if ACLY is causing the kidneys to produce and use less energy. This will be done by using mice with CKD and blocking the activity of ACLY using a drug to see if this will help the kidney create more energy for itself. The kidneys of the mice will be tested to see if the drug worked in increasing energy levels and if it prevented kidney scarring. A type of cell in the kidney, called tubular cells, makes up most of the kidney and requires a lot of energy to function. We performed experiments with tubular cells and gave them stressors, like those found in CKD, and ACLY-blockers to test if the energy levels are restored and if scarring was reduced. This study is important because there is no cure for CKD and many patients will eventually develop end-stage kidney disease, requiring dialysis or transplant. Research needs to be done to create new medications for those suffering from CKD. Current studies are testing ACLY-blocking drugs to treat heart disease. If our study is successful, this drug is well-positioned to be developed into a new treatment for CKD.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 127-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.

No description available.

Inorganic phosphate (Pi) plays a critical role in many biological structures and processes. However, Pi typically occurs at low concentrations, particularly in marine environments. In comparison, naturally occurring organophosphonates, which are characterized by a stable carbon-phosphorus (CP) bond, are frequently present at higher concentrations. Accordingly, bacteria have evolved different mechanisms for cleaving the CP bond of organophosphonates to liberate Pi for metabolic use. Two prominent enzyme pathways for catabolic cleavage of a CP bond are examined in this thesis. The first, called CP-lyase, is encoded by the phn operon that consists of 14 genes (phnCDEFGHIJKLMNOP). CP-lyase has long been of interest for its ability to degrade a wide array of organophosphonates through a homolytic CP bond cleaving reaction. A soluble protein complex consisting of PhnGHIJK was isolated from E. coli, suggesting that protein-protein interactions are important for CP bond cleavage. Intermediates of organophosphonate catabolism by E. coli CP-lyase were also detected and isolated, including -D-ribosyl-1,2-cyclic phosphate and N-acetylated aminoalkylphosphonates, 2-N-acetamidoethylphosphonate and 5’-phospho--D-ribosyl-1’-alkylphosphonates. The former compound was shown to be converted by the phosphodiesterase PhnP to -D-ribosyl-1-phosphate. It was also shown that PhnO is an aminoalkylphosphonate N-acetyl transferase and that N-acetylation by this enzyme is necessary for CP bond cleavage of 1-aminoalklyphosphonates. These results demonstrated that in addition to forming protein complexes, CP-lyase also comprises a catabolic pathway, with ribosylation of organophosphonates playing a key part in setting up the CP bond cleaving reaction. The second pathway examined in this thesis is comprised of marine bacterial enzymes PhnY and PhnZ and is specific for 2-aminoethylphosphonate. PhnY was shown to be an -ketoglutarate / Fe(II) dependent dioxygenase that hydroxylates the -carbon of 2-aminoethylphosphonate to form (R)-2-amino-1-hydroxyethylphosphonate. PhnZ was shown to be a novel Fe(II) dependent oxygenase that converts (R)-2-amino-1-hydroxyethylphosphonate to glycine and Pi. Site directed mutagenesis, kinetic analysis, reactions with substrate analogues, and X-ray crystallography examined the roles of active site residues and the di-iron active site. Additionally, a unique induced-fit mechanism was discovered which appears to synchronize substrate binding with activation of molecular oxygen. Overall these results show that PhnZ represents a new mechanism for catabolic cleavage of a CP bond. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-09-13 16:24:17.261

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.