published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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Peroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.

Class of 2006 Abstract / Objectives: To evaluate the efficacy of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, in reducing all-cause mortality and death due to rejection when administered to thoracic organ transplant patients. Methods: Using the following Medical Subject Heading (MeSH) terms and text words: hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, heart transplantation, and lung transplantation, the following data bases were searched: Cochrane Central Register of Controlled Trials (First Quarter 2006), Cochrane Database of Systematic Reviews (First Quarter 2006), Database of Abstracts and Reviews of Effects (First Quarter 2006), ACP Journal Club (1991to January/February 2006), International Pharmaceutical Abstracts (1970-February 2006), and Medline (1966 to February 2006) for English language reports. Three prospective randomized controlled trials (RCTs) and 3 retrospective observational studies were identified as using statins to reduce mortality and death due to fatal rejection in thoracic organ transplant patients. Results: Using all 6 studies (n= 1770 patients), statins decreased mortality by 77% (OR=0.23; [95% confidence interval 0.16-0.34] Z test, P<0.001). Sub-analysis using only RCT heart transplant data showed that statins decreased mortality by 69% (OR=0.31; [95% confidence interval 0.09-1.07] Z test, P<0.003). Sub-analysis using retrospective heart transplant data showed that statins decreased mortality by 75% (OR=0.25; [95% confidence interval 0.16-0.39] Z test, P<0.001). Retrospective lung transplant results (1 study) showed statins decreased mortality by 90% (OR=0.10; [95% confidence interval 0.03-0.34] Z test, P<0.001). Statins also significantly reduced death due to rejection (OR=0.22; [95% confidence interval 0.13-0.37]). Using all 6 studies (n= 1770 patients), statins decreased death due to rejection by 78%. Conclusions: In patients undergoing thoracic organ transplantation, statins significantly decrease all-cause mortality and death due to rejection. Therefore, statins should be routinely administered to these patients following transplant surgery.

Thesis advisor: Clare O'Connor / As organisms get older, the proteins in their cells also age, and as this happens, the amino acids that make up these proteins may become chemically modified and begin to lose their integrity. One example of an age-related modification occurs when the amino acid residue methionine is oxidized by a reactive oxygen species to methionine sulfoxide. Methionine sulfoxide reductase is an enzyme that repairs this damage to the protein by catalyzing a reaction that reduces methionine sulfoxide back to methionine. The fission yeast Schizosachharomyces pombe was used as the experimental model to study methionine sulfoxide reductase in vivo, taking advantage of the variety of tools available with which to study the organism. In S. pombe there are two genes encoding methionine reductase activities, msrA and msrB. The first goal of this project was to construct yeast strains in which the endogenous msrA and msrB genes had been inactivated. This was accomplished via homologous recombination reactions in which the msr genes were replaced with a selectable marker for biosynthesis of uracil (ura4+). After the construction and verification of the two knockout strains, the sensitivities of the strains to reactive oxygen species were tested. Both strains showed reduced resistance to oxidative stress. Future experiments will include more detailed analyses of the abilities of the strains to survive oxidative stress. Finally, the two knockout strains of yeast will be mated with one another in order to produce a double msr knockout, in order to examine the effects of a complete lack of methionine sulfoxide reductase activity on the organism. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

5a-Reductase 1 (5aR1) metabolises steroids such as glucocorticoids and androgens and is highly expressed in the livers of mice. Genetic disruption of 5aR1 leads to adverse metabolic consequences in mice and pharmacological inhibition in humans induces peripheral insulin resistance. I hypothesised that these effects are due to increased hepatic glucocorticoid action and firstly set up an experimental paradigm using A-348441, a liver-selective glucocorticoid receptor antagonist, to assess the contribution of hepatic glucocorticoid action. A-348441 was then utilised to assess whether changes in hepatic glucocorticoid signalling underpinned metabolic changes in: 1) a genetic model where the gene for 5aR1 has been disrupted, and 2) a pharmacological model using dutasteride, a dual 5aR1, R2 inhibitor. Previous work with A-348441 has demonstrated it can lower blood glucose levels in ob/ob mice. However, monogenic models of obesity are not fully representative of idiopathic obesity, which is commonly related to diet. Therefore, I utilised a mouse model of high fat dietary challenge to determine the effects of A-348441 in a more relevant model. High fat diet worsened metabolic indices such as body weight and weight gain, adipose tissue depot mass, fasting insulin and insulin response to a glucose challenge. A-348441 improved metabolic health of mice on high fat diet, preventing high fat-induced body weight gain, total white adipose depot weight gain and attenuating high fat-induced elevations in fasting plasma insulin, fasting glucose and insulin response to a glucose tolerance test. Importantly, hepatic glucocorticoid receptor antagonism did not change plasma corticosterone concentrations, indicating that glucocorticoid receptor antagonism was limited to the liver and thus demonstrating that hepatic glucocorticoid action plays a major role in high fat dietinduced metabolic phenotype. Using A-348441, I then went on to test the contribution of hepatic glucocorticoid action to the adverse metabolic phenotype in wild-type and 5aR1 knockout mice also under a high fat dietary challenge; two timescales were explored – 10 weeks with A- 348441 administered from the start and 6 months with A-348441 introduced after 5 months of high fat diet. 5aR1 knockout mice were overall more insulin resistant and had fattier livers than the wild-type mice at 10 weeks regardless of diet consumed. High fat diet overall worsened metabolic indices - increasing body weight, weight gain, adipose tissue depot mass, fasting insulin and insulin response to a glucose challenge in both genotypes and at both time points. Hepatic glucocorticoid receptor antagonism in 5aR1 knockout mice prevented high fat diet-induced metabolic consequences as expected in the 10-week high fat diet model, but not in the 6-month experiment; hyperinsulinaemia and weight gain were attenuated in the 10-week high fat diet model but not the 6-month high fat diet model, suggesting hepatic glucocorticoid receptor antagonism can prevent, but not reverse, high fat diet-induced metabolic consequences. However, A-348441 did not have a bigger effect on ameliorating the worsened metabolic state of the 5αR1 knockout mice. This suggests that increased hepatic glucocorticoid action does not underpin the adverse phenotype reported in the 5aR1 knockout mice. Dutasteride is a dual 5aR inhibitor prescribed to men for benign prostate hyperplasia or prostate cancer. I then recapitulated the human experiment where 5aR was pharmacologically inhibited and investigated the effects of dutasteride in mice. Inhibition of 5aRs in mice impaired insulin sensitivity, with increased insulin response to glucose tolerance test and also increased liver triglyceride levels; body weight, total adipose depot weight, fasting insulin, fasting glucose or glucose response to a glucose tolerance test were not changed by dutasteride. A-348441 reduced this hyperinsulinaemia but, as in other models, did not reduce the increased liver triglyceride levels. This suggests hepatic glucocorticoid action plays a substantial role in the development of insulin resistance caused by 5aR inhibition, but not in the development of hepatic steatosis. Therefore, adverse metabolic changes as a result of 5aR1 inhibition with dutasteride may be driven by altered hepatic glucocorticoid metabolism. Furthermore, metabolic changes caused by lifelong 5aR1 disruption are not responsive to short-term hepatic glucocorticoid receptor antagonism and altered androgen signalling may play a greater role. In conclusion, targeting the hepatic glucocorticoid receptor may be beneficial in restoring metabolic homeostasis in diet-induced obesity.

No description available.

This thesis reports structure-function assessments made using site-directed mutagenesis of the human enzyme 5alpha-reductase (5AR), an enzyme crucial for normal masculine development. These assessments utilised the differences between the two forms of the enzyme in affinity for testosterone and sensitivity to the competitive inhibitor Finasteride.Although first described in the 1950s, the enzyme has never been isolated and biochemical studies generated conflicting data. In some reports the enzyme was said to operate at a pH of 5.5 whereas others considered the pH optimum was more alkaline than this. Similarly, the affinity for testosterone was reported as being around 3muM and around 0.5muM and different sensitivities to the competitive inhibitor Finasteride also were reported. These differences were resolved in the period 1989 1991 by isolation of cDNAs coding for two forms of 5AR. These were termed 5AR1 and 5AR2. It is now known that 5AR1 operates at the more alkaline pH, has the lower affinity for testosterone and is the more resistant to Finasteride. 5AR1 is present to a greater extent in the periphery with 5AR2 predominating in the prostate.Isolation of the cDNAs coding for 5AR1 and 5AR2 and the differing characteristics of the two enzymes, have provided the means to investigate structure-function relationships using site-directed mutagenesis. This approach enables identification of residues important for the binding of substrate and inhibitor by the two forms of human 5AR.At the commencement of these studies the residues -AVFA- had been identified as comprising part of the substrate/inhibitor binding site of human 5AR1. As the analogous residues, -VSIV- of rat 5AR1 also had been shown to contribute to this binding, it had been suggested that the analogous residues -GALA- in human 5AR2 may form its substrate/inhibitor binding domain. There was however no ++ / experimental evidence to support this suggestion. Similarly the roles of particular residues identified as being involved in substrate/inhibitor binding by 5AR had not been subjected to significant study. Accordingly the experiments conducted were designed to address several unanswered questions. The most significant of these questions was are the residues -GALA- involved in substrate/inhibitor binding by human 5AR2 and if not what residues are in fact involved in this binding.Rat 5AR1 exhibits a different mechanism of Finasteride inhibition than does rat 5AR2and both of the human enzymes. This difference had been attributed to expression of a cysteine at position 146 in rat 5AR1 rather than the arginine expressed at the analogous position in rat 5AR2 and the human enzymes. The studies reported here for the mutation of the relevant arginine to cysteine do not substantiate the suggestion that cysteine 146 is responsible for the unique mechanism of inhibition by Finasteride shown by rat 5AR1.Although residues G34 and H231 in human 5AR2 had been shown important for substrate binding by this enzyme, the roles of these residues in inhibitor binding had not been examined nor had the roles of the analogous residues in human 5ARI (G39 and H236). Experiments designed to determine these factors demonstrated a requirement of both G34 and H231 for substrate and inhibitor binding by human 5AR2. The analogous residues in 5AR1 were however only required for the binding of inhibitor.The tetrapeptide -AVFA- had been shown to be involved in substrate/inhibitor binding by human 5AR1 but no assessment had been made of the relative roles of the individual amino acids comprising this tetrapeptide. Replacing residues in human 5AR1 with the corresponding residues from rat 5AR1 demonstrated a requirement in human 5AR1 of A26 and V27 for inhibitor but not substrate binding, possibly as a ++ / result of their conformational and electrostatic effects respectively. The branched chain residue V27 was found to be required for both substrate and inhibitor binding and the terminal alanine (A29) did not appear to play a significant role in the binding of either substrate or inhibitor. These studies also permitted an indirect assessment of the contribution of all four residues of the tetrapeptide -VSIV- to substrate binding by rat 5AR1 and showed that while the tripeptide -SIV- is important for inhibitor binding, the initial valine is not.As mentioned above the main impetus of these studies was to determine if residues -GALA- were involved in substrate/inhibitor binding by human 5AR2. Characterisation of mutants in which -GALA- in human 5AR2 was substituted with -AVFA- from 5AR1 and vice versa, showed these residues do not comprise the substrate/inhibitor binding site of human 5AR2.From studies of a chimeric constructs of human 5AR1 and 5AR2 residues involved in substrate/inhibitor binding by human 5AR2 were mapped to the first 40 amino acid residues. Mutations of 6 residue clusters showed the hexapeptide -ATLVAL(r15-20) in 5AR2 to contribute significantly to substrate/inhibitor binding. Further studies of this hexapeptide showed that residues -ATL- but not -VAL- form part of the substrate/inhibitor binding domain of human 5AR2.

Malaria and tuberculosis constitute two of the world’s deadliest infectious diseases. Together, they afflict over one third of the world’s population. Once thought of as one of a group of nearly vanquished diseases only 50 years ago, malaria and tuberculosis have experienced renewed prominence due to issues such as multi-drug resistance and a lack of responsiveness by the global community. Fatty acid biosynthesis has been shown to be an essential pathway to the causative organisms of malaria and tuberculosis. One integral component of the fatty acid biosynthesis pathway, enoyl acyl-carrier-protein (ACP) reductase, has repeatedly been validated as an appropriate drug target in other organisms. The 2.4 Å crystal structure of the enoyl-ACP reductase from the human parasite Plasmodium falciparum (PfENR) reveals a nucleotide-binding Rossmann fold, as well as the identity of several active site residues important for catalysis. The 2.43 Å crystal structure of PfENR bound with triclosan, a widely utilized anti-bacterial compound, provides new information concerning key elements of inhibitor binding. Applying knowledge attained from these initial crystal structures, several triclosan derivatives were synthesized, and subsequently PfENR:inhibitor co-crystal structures were determined to extend our knowledge of protein:inhibitor interactions within the active site. Additionally, the crystal structures of the enoyl-ACP reductase from the mouse parasite Plasmodium berghei (PbENR), in apo-form and in complex with triclosan, were refined to 2.9 Å and 2.5 Å resolution, respectively. These structures confirm the structural and active site conservation between the human and mouse parasite enoyl-ACP reductases, suggesting that utilizing a murine model for in vivo testing of promising inhibitors is viable. The 2.6 Å crystal structure of the enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) in complex with triclosan reveals a novel configuration of triclosan binding, where two molecules of triclosan are accommodated within the InhA active site. Finally, high-throughput screening approaches using enoyl acyl-carrier-protein reductases as the targets were utilized to identify new lead compounds for future generations of drugs. The 2.7 Å crystal structure of InhA bound with Genz-10850 confirms the value of this technique.

The 12-oxo-phytodienoic acid reductases (OPRs) are enzymes of the octadecanoid pathway which converts linolenic acid to a phytohormone, jasmonic acid. Bioinformatics analysis of ESTs and genomic sequences from available private and public databases revealed that the maize genome encodes eight different OPR genes. This number of maize OPR genes has been independently confirmed by Southern blot analysis and by mapping of individual OPR genes to maize chromosomes using oat maize chromosome addition lines. Survey of massively parallel signature sequencing (MPSS) assays revealed that transcripts of each OPR gene accumulate differentially in diverse organs of maize plants. This data suggested that individual OPR genes may have a distinct function in development. Similarly, RNA blot analysis revealed that distinct OPR genes are differentially regulated in response to stress hormones, wounding or pathogen infection. ZmOPR1 and ZmOPR2 appear to have important functions in defense responses to pathogens because they are transiently induced by salicylic acid (SA), chitooligosaccharides and by infection with Cochliobolus carbonum, Bipolaris maydis and Fusarium verticillioides and not by wounding. In contrast to these two genes, ZmOPR6 and ZmOPR7/8 are highly induced by wounding and treatments with wound-associated signaling molecules jasmonic acid, ethylene and abscisic acid. ZmOPR6 and ZmOPR7/8 are not induced by SA treatments or pathogen infections suggesting their specific involvement in wound-induced defense responses. Possible functions of specific OPR genes are discussed.