Jill Barber

Simultaneous Quantification of the Abundance of Several Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzymes in Human Liver Microsomes Using Multiplexed Targeted Proteomics

Cytochrome P450 (P450) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes mediate a major proportion of phase I and phase II metabolism of xenobiotics. In vitro-in vivo extrapolation (IVIVE) of hepatic clearance in conjunction with physiologically-based pharmacokinetics (PBPK) has become common practice in drug development. However, prediction of xenobiotic kinetics in virtual populations requires knowledge of both enzyme abundances and the extent to which these correlate. A multiplexed quantification concatemer (QconCAT) strategy was used in this study to quantify the expression of several P450 and UGT enzymes simultaneously and to establish correlations between various enzyme abundances in 24 individual liver samples (ages 27–66, 14 male). Abundances were comparable to previously reported values, including CYP2C9 (40.0 ± 26.0 pmol mg−1), CYP2D6 (11.9 ± 13.2 pmol mg−1), CYP3A4 (68.1 ± 52.3 pmol mg−1), UGT1A1 (33.6 ± 34.0 pmol mg−1), and UGT2B7 (82.9 ± 36.1 pmol mg−1), expressed as mean ± S.D. Previous reports of correlations in expression of various P450 (CYP3A4/CYP3A5*1/*3, CYP2C8/CYP2C9, and CYP3A4/CYP2B6) were confirmed. New correlations were demonstrated between UGTs [including UGT1A6/UGT1A9 (rs = 0.82, P < 0.0001) and UGT2B4/UGT2B15 (rs = 0.71, P < 0.0001)]. Expression of some P450 and UGT enzymes were shown to be correlated [including CYP1A2/UGT2B4 (rs = 0.67, P = 0.0002)]. The expression of CYP3A5 in individuals with *1/*3 genotype (n = 11) was higher than those with *3/*3 genotype (n = 10) (P < 0.0001). No significant effect of gender or history of smoking or alcohol use on enzyme expression was observed; however, expression of several enzymes declined with age. The correlation matrix produced for the first time by this study can be used to generate more realistic virtual populations with respect to abundance of various enzymes.

The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders.

Expression of Hepatic Drug-Metabolizing Cytochrome P450 Enzymes and Their Intercorrelations: A Meta-Analysis

Cytochrome P450 is a family of enzymes that catalyze reactions involved in the metabolism of drugs and other xenobiotics. These enzymes are therefore important in pharmacologic and toxicologic studies, and information on their abundances is of value in the process of scaling in vitro data to in vivo metabolic parameters. A meta-analysis was applied to data on the abundance of human hepatic cytochrome P450 enzymes in Caucasian adult livers (50 studies). Despite variations in the methods used to measure the abundance of enzymes, agreement between the studies in 26 different laboratories was generally good. Nonetheless, some heterogeneity was detected (Higgins and Thompson heterogeneity test). More importantly, large interindividual variability was observed in the collated data. Positive correlations between the expression levels of some cytochrome P450 enzymes were found in the abundance data, including the following pairs: CYP3A4/CYP3A5*1/*3 (Rs = 0.70, P < 0.0001, n = 52), CYP3A4/CYP2C8 (Rs = 0.68, P < 0.0001, n = 134), CYP3A4/CYP2C9 (Rs = 0.55, P < 0.0001, n = 71), and CYP2C8/CYP2C9 (Rs = 0.55, P < 0.0001, n = 99). These correlations can be used to demonstrate common genetic transcriptional mechanisms.

Cytochrome P450 pig liver pie: determination of individual cytochrome P450 isoform contents in microsomes from two pig livers using liquid chromatography in conjunction with mass spectrometry

The cytochrome P450 (P450) family of enzymes is a major player in the metabolism of therapeutic drugs available on the market, and the development of novel drugs has to take into account these enzymes in the fate of new drugs. Testing the pharmacokinetic behavior of new drugs in animals is a common part of the drug development process. Pigs are increasingly used for this purpose because of their similarity of enzymatic pattern to humans. In this study, adult Suffolk White pig liver microsomal samples were analyzed using mass-spectrometry-based techniques to identify and relatively quantify the porcine hepatic P450 enzymes. The total corrected microsomal protein content (milligrams of protein per gram of liver tissue) was estimated at 32.6 and 36.2 mg/g liver tissue in two samples, and the main identified liver P450 subfamilies were CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A. Label-free quantification was performed using the exponentially modified protein abundance index, and the highest abundance enzymes were CYP2A19 at 34% and CYP2D25 at 26% of the total identified drug-metabolizing P450 enzymes. The highest abundance subfamilies were CYP2A (34%), CYP2C (16%), CYP2D (26%), and CYP3A (14%). Moreover, primary sequence alignment was used to identify human homologs of the identified porcine P450s. Porcine CYP1A2 and CYP2E1 were shown to be equivalent to human CYP1A2 and CYP2E1, respectively. Porcine CYP2A19 has the highest sequence homology to human CYP2A6 and CYP2A13, and pig CYP2C33v4 and CYP2C49 are the porcine equivalent of human CYP2C9 and CYP2C18, respectively. Both identified pig CYP3A enzymes (CYP3A29 and CYP39) were highly homologous to CYP3A4/5.

Alternative fusion protein strategies to express recalcitrant QconCAT proteins for quantitative proteomics of human drug metabolizing enzymes and transporters

QconCAT is a tool for quantitative proteomics, consisting of an artificial protein, expressed from an artificial gene, made up of a concatenated string of proteotypic peptides selected from the proteins under study. Isotopically labeled QconCAT (usually containing (13)C6-arginine and (13)C6-lysine) provides a standard for each proteotypic peptide included in its sequence. In practice, some QconCAT proteins fail to express at sufficient levels for the purpose of quantitative analysis. Two complementary methods are presented to express recalcitrant QconCAT proteins intended to quantify human hepatic enzymes and transporters.

Protein expression of various hepatic uridine 5'-diphosphate glucuronosyltransferase (UGT) enzymes and their inter-correlations: a meta-analysis.

Avoiding cytochrome P450 (CYP) related drug interactions in the development of new drug candidates means that glucuronidation by uridine 5′‐diphosphate glucuronosyltransferase (UGT) enzymes is expected to become a more prominent pathway in the metabolism of new drug candidates designed by pharmaceutical companies. Therefore, determining the abundance and activity of these enzymes is of value in the process of scaling in vitro data to in vivo metabolic parameters. Many of the studies involving the measurement of UGTs were conducted with too few samples, which did not provide a good indication of population values and the level of variability. Meta‐analysis is used in the current study to combine all reported values (eight studies that used LC‐MS isotope‐labelled standard targeted quantitative methods), detect inconsistencies between the various datasets and describe correlations of expression between the quantified UGT enzymes. Some heterogeneity was observed between studies, especially in the UGT1A4, 2B7 and 2B10 datasets. However, in the absence of information on the inter‐laboratory consistency of assays, it is difficult to assign these differences to the heterogeneity of the samples. Large inter‐individual variability was observed in the collated data across this family of enzymes. Positive correlations between the expression levels of certain UGT enzymes were found in the collated data. These included the pairs: UGT1A4/2B4 (rs = 0.71, p < 0.0001, n = 82), UGT2B4/2B15 (rs = 0.63, p < 0.0001, n = 83), UGT2B7/2B15 (rs = 0.81, p < 0.0001, n = 99). These correlations can be explained by common regulatory mechanisms involved in the expression of these proteins. Copyright

Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum

Glutathione S‐transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium‐tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom‐bound deuteriums (in addition to the carbon‐bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light‐scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar Km to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms

In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.

Quantification of the Proteins of the Bacterial Ribosome Using QconCAT Technology

The bacterial ribosome is a complex of three strands of RNA and approximately 55 proteins. During protein synthesis, the ribosome interacts with other proteins, numbered in the hundreds, forming some stable and some transient complexes. The stoichiometries of these complexes and of partially assembled ribosomes are often unknown. We describe the development of a flexible standard for the determination of stoichiometries of ribosomal particles and complexes. A core QconCAT, an artificial protein consisting of concatenated signature peptides derived from the ribosomal proteins L2, L4, L13, S4, S7, and S8, was developed. The core QconCAT DNA construct incorporates restriction sites for the insertion of cassettes encoding signature peptides from additional proteins under study. Two cassettes encoding signature peptides from the remaining 30S and 50S ribosomal proteins were prepared, and the resulting QconCATs were expressed, digested, and analyzed by mass spectrometry. The majority of Escherichia coli ribosomal proteins are small and basic; therefore, tryptic digestion alone yields insufficient signature peptides for quantification of all of the proteins. The ribosomal QconCATs therefore rely on a dual-enzyme strategy: endoproteinase Lys-C digestion and analysis followed by trypsin digestion and further analysis. The utility of technology was demonstrated by a determination of the effect of gentamicin on the protein composition of the E. coli ribosome.

Free and bound state structures of 6-O-methyl homoerythromycins and epitope mapping of their interactions with ribosomes.

The solution and solid state conformations of several 6-O-methyl homoerythromycins 1-4 were studied using a combination of X-ray crystallography, NMR spectroscopy and molecular modelling calculations. In the solid state 1 was found to exist as the two independent molecules with similar structures termed 3-endo-folded-out. In solution a significant conformational flexibility was noticed especially in the C2 to C5 region. The compounds 1 and 2 unlike 14-membered macrolides adopted the 3-endo-folded-out conformation while 3 and 4 existed in the classical folded-out conformation. TrNOESY and STD experiments showed that 1 and 2 bound to the Escherichia coli ribosome while 3 and 4, lacking the cladinose sugar, did not exhibit binding activities, this being in accordance with biochemical data. The bound conformations were found to be very similar to the free ones, some small differences were observed and discussed. The STD experiments provided evidence on binding epitopes. The structural parts of 1 and 2 in close contact with ribosome were similar, however the degree of saturation transfer was higher for 2. The differences between tr-NOE data and STD enhancements in 1 and 2 arouse as a consequence of structural changes upon binding and a closer proximity of 2 to the ribosome surface. An understanding of the molecular mechanisms involved in the interaction of macrolides with ribosomes can help in developing strategies aiming at design of potential inhibitors.