A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.

Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...

<p>Cytochrome b5 heme binding domain 2 (CYB5D2) is a heme binding protein that was initially identified for its ability to attenuate the function of the PTEN tumor suppressor gene. CYB5D2 sustains ectopic PTEN expression in U87 cells, and can also confer survival from serum starvation in NIH3T3 cells. An antibody was generated to the carboxyl terminus of CYB5D2 to detect endogenous protein expression. The highest expression of CYB5D2 protein is in neural cancer cell lines. CYB5D2 is weakly expressed in breast and kidney cancer cell lines, and moderately expressed in prostate cancer cell lines. To investigate the role of the heme binding domain in CYB5D2, a conserved aspartic acid (D86) within this domain was mutated to glycine, and this was characterized as being unable to bind heme. CYB5D2(D86G) displayed a loss of function compared to wild-type CYB5D2. To study the loss of expression of CYB5D2, stable CYB5D2 shRNA was achieved in HeLa and Huh7 cells. While ectopic CYB5D2 inhibited HeLa cell proliferation and growth in soft agar, CYB5D2(D86G) expression and CYB5D2 shRNA increased cell proliferation and soft agar growth. While ectopic CYB5D2 conferred survival from chemotherapeutic drugs in HeLa cells, CYB5D2(D86G) and CYB5D2 shRNA cells were susceptible to drug treatments. CYB5D2 inhibits SREBP signalling, which requires its heme binding ability. Using cyclohexamide treatments, CYB5D2 stabilized ectopic Insig1, while CYB5D2(D86G) destabilized ectopic Insig1. CYB5D2 shRNA reduced endogeneous CYP51A1 (lanosterol demethylase) and Insig1 protein levels, and increased the susceptibility of HeLa cells to mevalonate treatments. Furthermore, CYB5D2 shRNA HeLa cells displayed reduced CYP3A4 activity, a cytochrome P450 enzyme involved in drug metabolism. CYB5D2 binds to cytochrome P450 reductase (POR), while CYB5D2(D86G) cannot. CYB5D2 co-immunoprecipitates with endogenous POR under serum-free conditions in HeLa and Huh7 cells, while CYB5D2(D86G) cannot. Collectively, CYB5D2 is a POR interacting protein, which regulates CYP51A1 and CYP3A4 activity.</p> / Doctor of Philosophy (Medical Science)

No / A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

The cytochrome P450 enzyme system is a multicomponent electron-transfer chain composed of a haem-containing monooxygenase cytochrome P450 (CYP) and one or more redox partners. Eukaryotic CYPs and their redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are involved in many biological processes. Each protein has one N- terminal membrane anchor domain for location within the endoplasmic reticulum (ER). In mammals, CYPs and CPR are especially abundant in liver cells, where they play important roles in the metabolism of steroids, fatty acids, and xenobiotic compounds including numerous drugs of pharmaceutical importance. Incorporation into lipid membranes is an important aspect of CYP and CPR function, influencing their kinetic properties and interactions. In this thesis, soluble nanometer-scale phospholipid bilayer membrane discs, "nanodiscs", were used as a reconstitution system to study the influence of lipid membrane composition on the activities of the abundant human CYP3A4 and human CPR. Both enzymes were expressed and purified from bacteria, and assembled into functionally active membrane-bound complexes in nanodiscs. Nanodisc assembly was assessed by a combination of native and denaturing gel electrophoresis, and a fluorimetric assay was developed to study CYP3A4 reaction kinetics using 7-benzyloxyquinoline as substrate. Kinetic properties were investigated with respect to different lipid membrane compositions: phosphatidyl choline; a synthetic lipid mixture resembling the ER; and natural lipids extracted from liver microsomes. Full activity of the CYP3A4 system, with electron transfer from NADPH via CPR, could only be reconstituted when both CYP3A4 and CPR were membrane-bound within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated seperately into naodiscs then mixed together, or when soluble forms of CPR were mixed with pre-assembled CYP3A4-nanodiscs. Thus, assembly of the two proteins within the same membrane was shown to be essential for the function of the CPR-CYP3A4 electron transfer system. Comparison of the reaction kinetics in different membrane compositions revealed liver microsomal lipid to have an enhancing effect both on the activity of the assembled CPR-CYP3A4 nanodisc complex, and on the activity of CPR alone incorporated in nanodiscs, when compared either to the synthetic lipid mixture or to phosphatidyl choline alone. Thus, natural lipids appear to possess properties or include components important for the catalytic function of the CYP system, which are absent from synthetic lipid. Input of electrons, measured by NADPH consumption, exceeded product formation rate by the CPR-CYP3A4 complex in nanodiscs, indicating "leakage" in the electron flow, possibly due to uncoupling of the two enzymes. Uncoupling was shown to occur by developing a novel fluorimetric method using the dye MitSOX to detect superoxide production. The significance of this, and to what extent control of coupling could be a natural means of regulation of the CPR-CYP system, remains to be determined. Thus, phospholipid bilayer nanodiscs prove a powerful tool to enable detailed analysis of the reaction kinetics of membrane-reconstituted CPR-CYP systems, and to allow pertinent questions to be addressed concerning the integral significance of the membrane environment.

La NADPH cytochrome P450 réductase (CPR) est une flavoprotéine multidomaines appartenant à la famille des diflavines réductases et un des composants essentiels du système redox des cytochromes P450. La CPR est formée de deux domaines catalytiques contenant des groupements prosthétiques FAD et FMN et d'un domaine de connexion. Le domaine FAD reçoit deux électrons du NADPH et les transfère un par un au domaine FMN, qui, à son tour, les transfère aux accepteurs. Le transfert d’électron du FMN vers les accepteurs nécessite un déplacement du domaine FMN par rapport au reste de la molécule. Au fils des années, les études structurales menées sur la CPR ont mis en évidence la réorganisation structurale et l’arrangement des domaines dans cette protéine. Cependant, les résultats de ces analyses ne fournissent pas d’informations concernant la vitesse à laquelle les mouvements des domaines de la CPR s’effectuent et n’incluent toujours pas les paramètres qui induisent le changement conformationnel ainsi que l'influence de ces changements sur l’activité catalytique de la CPR.Le projet de cette thèse a consisté à apporter de nouveaux éléments de compréhension sur la relation entre les changements conformationnels de la CPR et son cycle catalytique. La première partie de ce travail a porté sur le développement de stratégies de préparation au marquage des domaines catalytiques de la CPR, destinés à l’étude dynamique de cette protéine par le FRET. Différentes stratégies ont été envisagées parmi lesquelles l’incorporation de p-acétyle phénylalanine sur des positions définies dans la CPR. La deuxième partie de ce travail est consacrée à l’étude dynamique de la CPR via des techniques de RMN et SAXS combinées à des approches biochimiques. Les expériences menées ont permis de caractériser en solution et en absence de NaCl, la présence d’un état rigide, globulaire en conformation fermée dans laquelle les domaines FMN et FAD sont maintenus « verrouillés » par des interactions à l’interface entre ces deux domaines. L’augmentation de la concentration en NaCl permet une transition de cet état « verrouillé » vers un état plus ouvert pour lequel il n’y a plus d’interface entre les domaines FAD et FMN. L’état « déverrouillé » de la CPR correspond à un équilibre dynamique entre un ensemble de conformations en échange rapide. Cet équilibre est contrôlé par la force ionique et l’activité catalytique de la CPR est maximale lorsque les états verrouillés et déverrouillés sont également peuplés. Le modèle cinétique proposé par nos études a permis de mettre en évidence un lien direct entre la dynamique des domaines et l’activité du transfert d’électron au cours de cycle catalytique de la CPR. / NADPH cytochrome P450 reductase (CPR) is a multidomain flavoprotein that belongs to the diflavines reductase family. It is an essential component of redox system delivering electrons for cytochrome P450. CPR is composed of two catalytic domains containing FAD and FMN prosthetic groups and a connecting domain. FAD domain receives two electrons from NADPH and transfers them one by one to the FMN domain, which in turn transfers them to the acceptor. The electron transfer from FMN to the acceptor requires a large domain movement. Over the years, structural studies of CPR have highlighted the reorganization and arrangement of domains in this protein. However, the results of these analyses do not provide any information about how fast the domains movements takes place in CPR, and do not always include the parameters that induce conformational change as well as influence of those changes on the catalytic activity of the CPR. This thesis aims to bring new elements of comprehension on the relationship between conformational changes in CPR and its catalytic cycle. The first part of the work concerned the development of strategies to label the catalytic domains of CPR, a prerequisite for the dynamic study of this protein by FRET. Different strategies have been proposed including the incorporation of p-acetyl phenylalanine at defined positions in CPR. The second part of this work is devoted to the dynamic study of CPR through a combined SAXS, NMR and biochemical approaches. The experiments conducted allowed to characterize the presence of a rigid and globular state of closed conformation for CPR, in solution and in the absence of NaCl. In this conformation the FMN and FAD domains are kept "locked" by interface interactions between these two domains. Increasing the NaCl concentration permits the transition from the "locked" stats to an open conformation in which there is no more interface between the FAD and FMN domains. The "unlocked" state of CPR is a dynamic equilibrium between ensembles of conformations in fast exchange. This equilibrium is controlled by the ionic strength and CPR presents its maximum catalytic activity when the locked and unlocked states are equally populated. We proposed a kinetic model which allows demonstrating a direct link between the domain movement and electron transfer activity during the catalytic cycle of CPR.