MYLK pathogenic variants aortic disease presentation, pregnancy risk, and characterization of pathogenic missense variants.

PURPOSE: Heritable thoracic aortic disease can result from null variants in MYLK, which encodes myosin light-chain kinase (MLCK). Data on which MYLK missense variants are pathogenic and information to guide aortic disease management are limited. METHODS: Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed. RESULTS: Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm repair or dissection); the majority of these events (87%) were aortic dissections. Aortic diameters were minimally enlarged at the time of dissection in many cases. Time-to-aortic-event curves showed that missense pathogenic variant (PV) carriers have earlier-onset aortic events than null PV carriers. An MYLK missense variant segregated with aortic disease over five generations but decreases MYLK kinase acitivity marginally. Functional Assays fail to identify all pathogenic variants in MYLK. CONCLUSION: These data further define the aortic phenotype associated with MYLK pathogenic variants. Given minimal aortic enlargement before dissection, an alternative approach to guide the timing of aortic repair is proposed based on the probability of a dissection at a given age.

Variants of Unknown Significance in Genes Associated with Heritable Thoracic Aortic Disease Can Be Low Penetrant "Risk Variants".

Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of premature deaths if individuals at risk can be identified. Individuals with early-onset aortic dissections without a family history or syndromic features have an increased burden of rare genetic variants of unknown significance (VUSs) in genes with pathogenic variants for heritable thoracic aortic disease (HTAD). We assessed the role of VUSs in the development of disease using both in vitro enzymatic assays and mouse models. VUSs in LOX and MYLK identified in individuals with acute aortic dissections were assayed to determine whether they disrupted enzymatic activity. A subset of VUSs reduced enzymatic activity compared to the wild-type proteins but less than pathogenic variants. Additionally, a Myh11 variant, p.Arg247Cys, which does not cause aortic disease in either humans or mice, was crossed with the Acta2-/- mouse, which has aortic enlargement with age while Acta2+/- mice do not. Acta2+/-Myh11R247C/R247C mice have aortic dilation by 3 months of age without medial degeneration, indicating that two variants not known to cause disease do lead to aortic enlargement in combination. Furthermore, the addition of Myh11R247C/R247C to the Acta2-/- mouse model accelerates aortic enlargement and increases medial degeneration. Therefore, our results emphasize the need for a classification system for variants in Mendelian genes that goes beyond the 5-tier system of pathogenic, likely pathogenic, VUS, likely benign, and benign, and includes a designation for low-penetrant "risk variants" that trigger disease either in combination with other risk factors or in a stochastic manner.

Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function.

The inhibition of platelet aggregation is key to preventing conditions such as myocardial infarction and ischemic stroke. Aspirin is the most widely used drug to inhibit platelet aggregation. Aspirin absorption can be improved further to increase its permeability across biologic membranes via esterification or converting the carboxylic acid to an anhydride. There are several reports indicating that ω-3 and ω-6 fatty acids such as linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) separately inhibit platelet aggregation. Herein, we synthesize anhydride conjugates of aspirin with linoleic acid, EPA, and DHA to form aspirin anhydrides that are expected to have higher permeability across cellular membranes. These aspirin-fatty acid anhydrides inhibited platelet aggregation in washed human platelets and platelet-rich plasma in a dose-dependent manner. In particular, the aspirin-DHA anhydride displayed similar effectiveness to aspirin. Platelet aggregation studies conducted in the presence of various platelet agonists indicated that the aspirin-lipid conjugates act through inhibition of the cyclooxygenase (COX)-thromboxane synthase (TXAS) pathway. Hence, we performed detailed biochemical studies using purified COX-1 as well as TXAS stabilized in nanoscale lipid bilayers of nanodiscs to confirm results from the platelet aggregation studies. We show that although all of the aspirin conjugates act through the COX-TXAS pathway by inhibiting COX-1, the parent fatty acids do not act via this pathway. Finally, we studied the hydrolysis of these compounds in buffer and human plasma, and we demonstrate that all of the aspirin-fatty acid conjugates hydrolyze to the parent molecules aspirin and fatty acid in a controlled manner.

Genetic variation in UGT genes modify the associations of NSAIDs with risk of colorectal cancer: colon cancer family registry.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal neoplasia. Previous studies have reported that polymorphisms in NSAID-metabolizing enzymes central to NSAID metabolism including UDP-glucuronosyltransferases (UGT) and cytochrome P450 (CYP) 2C9 may modify this protective effect. We investigated whether 35 functionally relevant polymorphisms within CYP2C9 and UGT genes were associated with colorectal cancer risk or modified the protective effect of NSAIDs on colorectal cancer susceptibility, using 1,584 colorectal cancer cases and 2,516 unaffected sibling controls from the Colon Cancer Family Registry. A three-SNP genotype in UGT1A6 (G-A-A; Ala7-Thr181-Arg184) and the Asp85 variant in UGT2B15 increased the risk of colorectal cancer (OR 3.87; 95% CI 1.04-14.45 and OR 1.34; 95% CI 1.10-1.63, respectively). We observed interactions between UGT1A3 Thr78Thr (A>G) and NSAID use (P-interaction = 0.02), a three-SNP genotype within UGT2B4 and ibuprofen use (P-interaction = 0.0018), as well as UGT2B15 Tyr85Asp (T>G) and aspirin use (P-interaction = 0.01). The interaction with the UGT2B4 and the UGT2B15 polymorphisms were noteworthy at the 25% FDR level. This study highlights the need for further pharmacogenetic studies to identify individuals who might benefit from NSAID use as part of developing effective strategies for prevention of colorectal neoplasia. © 2014 Wiley Periodicals, Inc.

Phospholipase A2G1B polymorphisms and risk of colorectal neoplasia.

Pancreatic phospholipase A2, product of PLA2G1B, catalyzes the release of fatty acids from dietary phospholipids.Diet is the ultimate source of arachidonic acid in cellular phospholipids, precursor of eicosanoid signaling molecules, linked to inflammation, cell proliferation and colorectal carcinogenesis. We evaluated the association of PLA2G1B tagging single-nucleotide polymorphisms with colorectal neoplasia risk. A linkage-disequilibrium-based tagSNP algorithm (r(2)=0.90, MAF≥4%) identified three tagSNPs. The SNPs were genotyped on the Illumina platform in three population-based, case-control studies: colon cancer (1424 cases/1780 controls); rectal cancer (583/775); colorectal adenomas (485/578). Evaluating gene-wide associations, principal-component and haplotype analysis were conducted, individual SNPs were evaluated by logistic regression. Two PLA2G1B variants were statistically significantly associated with reduced risk of rectal cancer (rs5637, 3702 G>A Ser98Ser, p-trend=0.03; rs9657930, 1593 C>T, p-trend=0.01); principal component analysis showed that genetic variation in the gene overall was statistically significantly associated with rectal cancer (p=0.02). NSAID users with the rs2070873 variant had a reduced rectal cancer risk (P-inter=0.02). Specific associations were observed with tumor subtypes (TP53/KRAS). The results suggest that genetic polymorphisms in PLA2G1B affect susceptibility to rectal cancer.

COX-1 (PTGS1) and COX-2 (PTGS2) polymorphisms, NSAID interactions, and risk of colon and rectal cancers in two independent populations.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) target the prostaglandin H synthase enzymes, cyclooxygenase (COX)-1 and COX-2, and reduce colorectal cancer risk. Genetic variation in the genes encoding these enzymes may be associated with changes in colon and rectal cancer risk and in NSAID efficacy. METHODS: We genotyped candidate polymorphisms and tag SNPs in PTGS1 (COX-1) and PTGS2 (COX-2) in a population-based case­control study (Diet, Activity and Lifestyle Study, DALS) of colon cancer (n = 1,470 cases/1,837 controls) and rectal cancer (n = 583/775), and independently among cases and controls from the Colon Cancer Family Registry (CCFR; colon n = 959/1,535, rectal n = 505/839). RESULTS: In PTGS2, a functional polymorphism (-765G[C; rs20417) was associated with a twofold increased rectal cancer risk (p = 0.05) in the DALS. This association replicated with a significant nearly fivefold increased risk of rectal cancer in the CCFR study (ORCC vs. GG = 4.88; 95 % CI 1.54­15.45; ORGC vs. GG = 1.36; 95 %CI 0.95­1.94). Genotype­NSAID interactions were observed in the DALS for PTGS1 and rectal cancer risk and for PTGS2 and colon cancer risk, but were no longer significant after correcting for multiple comparisons and did not replicate in the CCFR. No significant associations between PTGS1 polymorphisms and colon or rectal cancer risk were observed.

IκBKß and NFκB1, NSAID use and risk of colorectal cancer in the Colon Cancer Family Registry.

The NFκB-signaling pathway regulates cell proliferation and inflammation. Activation of the pathway is implicated in the etiology of colorectal cancer (CRC). NSAIDs may reduce CRC risk partially through a nuclear factor-kappa B (NFκB)-dependent pathway. In this study, we investigated associations between 34 NFκB1 and 8 IκBKß tagSNPs and CRC risk and examined interactions with non-steroidal anti-inflammatory drug (NSAID) use. Using conditional logistic regression, we investigated these associations among 1584 incident CRC cases and 2516 sibling controls from the Colon Cancer Family Registry. Three IκBKß SNPs were associated with a statistically significant lower colorectal or colon cancer risk: rs9694958 (A>G intron 5) (colorectal: OR(hzv) = 0.26(0.07-0.99), P(trend) = 0.048, P(adj) = 0.25), rs10958713 (A>C intron 19) (colon: OR(hzv) = 0.62(0.42-0.92), P(trend) = 0.005, P(adj) = 0.03) and rs5029748 (C>A intron 2) (colon: OR(het) = 0.72(0.56-0.91), P(trend) = 0.01, P(adj) = 0.08). We replicated trends associated with NFκB1 and IκBKß variants identified in a previous study (rs4648110 (T>A intron 22), rs13117745 (G>A intron 5) and rs3747811 (T>A intron 1)). IκBKß's rs6474387 (C>T intron 20) and rs11986055 (A>C intron 2) showed substantially lower colon cancer risk among current NSAID users (P(interaction) = 0.01 and P(interaction) = 0.045, respectively), whereas NFκB1's rs230490 (G>A 5' (outside UTR)) and rs997476 (C>A 3' (outside UTR)) showed higher CRC risk among current NSAID users (P(interaction) = 0.01 and P(interaction) = 0.03, respectively). These findings suggest that variants in NFκB1 and IκBKß are associated with CRC risk and NSAIDs may function partially through an NFκB-dependent pathway. The SNPs identified here should be considered for future functional studies and may be useful in designing a pharmacogenetic approach to preventive NSAID use.

PTGS1, PTGS2, ALOX5, ALOX12, ALOX15, and FLAP SNPs: interaction with fatty acids in colon cancer and rectal cancer.

Dietary polyunsaturated fatty acids (PUFAs) can be converted to prostaglandins and leukotrienes. Oxygenation of omega-6 PUFAs generally results in the production of pro-inflammatory mediators, whereas oxygenated products of omega-3 (n-3) PUFAs generally have lower inflammatory activity. We hypothesize that elevated n-3 PUFA intakes from fish are associated with lower risk of colorectal cancer among those with genetic variants that result in higher levels of pro-inflammatory mediators. In population-based case-control studies of colon (case n = 1,574) and rectal cancer (case n = 791) and disease-free controls (n = 2,969), we investigated interactions between dietary fatty acid intake and 107 candidate polymorphisms and tagSNPs in PTGS1, PTGS2, ALOX12, ALOX5, ALOX15, and FLAP. The two studies used an identical genotyping protocol. We observed interactions and statistically significant increases in colon cancer risk for low docosahexaenoic acid intake among those with the PTGS1 rs10306110 (-1,053 A > G) variant genotypes (OR = 1.6, 95 % confidence interval = 1.1-2.3, adj. p = 0.06) and rectal cancer risk for low total fat intake among those with the variant PTGS1 rs10306122 (7,135 A > G) (OR(vs.wt) = 1.80, 1.02-2.99; adj. p = 0.08). The ALOX15 rs11568131 (10,339 C > T) wild type in combination with a high inflammation score (low EPA intake, high AA intake, no regular NSAID use, high BMI, smoking) was associated with increased colon cancer risk (OR = 2.28, 1.7-3.07). Rectal cancer risk was inversely associated with a low inflammation score among PTGS2 rs4648276 (3,934 T > C) variant allele carriers (OR = 0.49, 0.25-0.75). Overall, these data provide some modest evidence for interactions between dietary fat intake and genetic variation in genes involved in eicosanoid metabolism and colorectal cancer risk.

Functional analysis of human thromboxane synthase polymorphic variants.

BACKGROUND: Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA2 generation. OBJECTIVES: The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. METHODS: Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. RESULTS: All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 µmol/l PGH2 (wild-type value: 32 µmol/l PGH2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose Vmax/Km ratio was just 27% of the wild-type value. CONCLUSION: The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.

Decreased cyclooxygenase inhibition by aspirin in polymorphic variants of human prostaglandin H synthase-1.

OBJECTIVES: Aspirin (ASA), a major antiplatelet and cancer-preventing drug, irreversibly blocks the cyclooxygenase (COX) activity of prostaglandin H synthase-1 (PGHS-1). Considerable differences in ASA effectiveness are observed between individuals, and some of this variability may be due to PGHS-1 protein variants. Our overall aim is to determine which, if any, of the known variants in the mature PGHS-1 protein lead to functional alterations in COX catalysis or inhibition by ASA. The present study targeted four PGHS-1 variants: R53H, R108Q, L237M, and V481I. METHODS: Wild-type human PGHS-1 and the four polymorphic variants were expressed as histidine-tagged, homodimeric proteins in insect cells using baculovirus vectors, solubilized with a detergent, and purified by affinity chromatography. The purified proteins were characterized in vitro to evaluate COX and peroxidase (POX) catalytic parameters and the kinetics of COX inhibition by ASA and NS-398. RESULTS: Compared with the wild type, several variants showed a higher COX/POX ratio (up to 1.5-fold, for R108Q), an elevated arachidonate Km (up to 1.9-fold, for R108Q), and/or a lower ASA reactivity (up to 60% less, for R108Q). The decreased ASA reactivity in R108Q reflected both a 70% increase in the Ki for ASA and a 30% decrease in the rate constant for acetyl group transfer to the protein. Computational modeling of the brief ASA pulses experienced by PGHS-1 in circulating platelets during daily ASA dosing predicted that the 60% lower ASA reactivity in R108Q yields a 15-fold increase in surviving COX activity; smaller, approximately two-fold increases in surviving COX activity were predicted for L237M and V481I. NS-398 competitively inhibited COX catalysis of the wild type (Ki=6 µmol/l) and inhibited COX inactivation by 1.0 mmol/l ASA in both the wild type (IC50=0.8 µmol/l) and R108Q (IC50=2.1 µmol/l). CONCLUSION: Of the four PGHS-1 variants examined, R108Q exerts the largest functional effects, with evidence for impaired interactions with a COX substrate and inhibitors. As Arg108 is located on the protein surface and not in the active site, the effects of R108Q suggest a novel, unsuspected mechanism for the modulation of the PGHS-1 active site structure. The lower intrinsic ASA reactivity of R108Q, V481I, and L237M, combined with the rapid hydrolysis of ASA in the blood, suggests that these variants decrease the antiplatelet effectiveness of the drug. These PGHS-1 variants are uncommon but ASA is used widely; hence, a considerable number of individuals could be affected. Further examination of these and other PGHS-1 variants will be needed to determine whether PGHS-1 genotyping can be used to personalize anti-COX therapy.

Glutathione peroxidase tagSNPs: associations with rectal cancer but not with colon cancer.

Glutathione peroxidases (GPXs) are selenium-dependent enzymes that reduce and, thus, detoxify hydrogen peroxide and a wide variety of lipid hydroperoxides. We investigated tagSNPs in GPX1-4 in relation to colorectal neoplasia in three independent study populations capturing the range of colorectal carcinogenesis from adenoma to cancer. A linkage-disequilibrium (LD)-based tagSNP selection algorithm (r(2) ≥ 0.90, MAF ≥ 4%) identified 21 tagSNPs. We used an identical Illumina platform to genotype GPX SNPs in three population-based case-control studies of colon cancer (1,424 cases/1,780 controls), rectal cancer (583 cases/775 controls), and colorectal adenomas (485 cases/578 controls). For gene-level associations, we conducted principal component analysis (PCA); multiple logistic regression was used for single SNPs. Analyses were adjusted for age, sex, and study center and restricted to non-Hispanic white participants. Analyses of cancer endpoints were stratified by molecular subtypes. Without correction for multiple testing, one polymorphism in GPX2 and three polymorphisms in GPX3 were associated with a significant risk reduction for rectal cancer at α = 0.05, specifically for rectal cancers with TP53 mutations. The associations regarding the three polymorphisms in GPX3 remained statistically significant after adjustment for multiple comparisons. The PCA confirmed an overall association of GPX3 with rectal cancer (P = 0.03). No other statistically significant associations were observed. Our data provide preliminary evidence that genetic variability in GPX3 contributes to risk of rectal cancer but not of colon cancer and thus provide additional support for differences in underlying pathogenetic mechanisms for colon and rectal cancer.

Genetic variability in EGFR, Src and HER2 and risk of colorectal adenoma and cancer.

The EGFR signaling pathway is involved in carcinogenesis at multiple sites, particularly colorectal cancer, and is a target of colorectal cancer chemotherapy. EGFR signaling is linked to pro-carcinogenic mechanisms, including cell proliferation, survival, angiogenesis, and more recently prostaglandin synthesis. Genetic variability in this pathway has not yet been studied in relation to colorectal carcinogenesis. In three case-control studies of colorectal adenoma (n=485 cases/578 controls), colon cancer (n=1424 cases/1780 controls) and rectal cancer (n=583 cases/775 controls), we investigated associations between candidate SNPs, tagSNPs and haplotypes in EGFR signaling (EGFR, Src, and HER2) and risk. We also examined associations with tumor subtypes: TP53 and KRAS2 mutations, CpG island methylator phenotype, and microsatellite instability. All three studies were genotyped using an identical Illumina GoldenGate assay, allowing thorough investigation of genetic variability across stages and locations of colorectal neoplasia. The EGFR tagSNP 142572T>C (rs3752651) CC genotype was associated with a suggested increased risk for both colon (OR: 1.40; 95% CI: 1.00-1.96; p-trend=0.04) and rectal cancer (OR: 1.39; 95% CI: 0.81-2.41; p-trend=0.65). In tumor subtype analyses, the association was limited to TP53-mutated colon tumors. Using the Chatterjee 1 df Tukey test to assess gene-gene interactions, we observed a statistically significant (p<0.01) interaction between SNPs in EGFR and Src for colorectal adenoma risk. The association with EGFR 142572 should be investigated in additional studies and the significant gene-gene interaction between EGFR and Src in relation to adenoma risk suggests that these two genes are jointly affecting early stages in colorectal carcinogenesis and requires further follow-up.

High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system.

Human duodenal cytochrome b (Dcytb) is a transmembrane hemoprotein found in the duodenal brush border membrane and in erythrocytes. Dcytb has been linked to uptake of dietary iron and to ascorbate recycling in erythrocytes. Detailed biophysical and biochemical characterization of Dcytb has been limited by difficulties in expressing sufficient amounts of functional recombinant protein in yeast and insect cell systems. We have developed an Escherichia coli Rosetta-gami B(DE3) cell system for production of recombinant His-tagged human Dcytb with a yield of ∼26 mg of purified, ascorbate-reducible cytochrome per liter of culture. The recombinant protein is readily solubilized with n-dodecyl-ß-D-maltoside and purified to electrophoretic homogeneity by one-step chromatography on cobalt affinity resin. The purified recombinant Dcytb has a heme to protein ratio very close to the theoretical value of 2 and retains functional reactivity with ascorbate, as assessed by spectroscopic and kinetic measurements. Ascorbate showed a marked kinetic selectivity for the high-potential heme center over the low-potential heme center in purified Dcytb. This new E. coli expression system for Dcytb offers ∼7-fold improvement in yield and other substantial advantages over existing expression systems for reliable production of functional Dcytb at levels suitable for biochemical, biophysical and structural characterization.

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2.

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O(2). The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.

Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561.

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.

Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects.

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using unlabeled AA and several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d(2)-AA and 10, 10, 13,13-d(4)-AA. The primary KIE for steady-state cyclooxygenase catalysis, (D)k(cat), ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9-2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes slow formation of a pentadienyl AA radical and rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of lipoxygenase indicates very different mechanism of hydrogen transfer.

Cyclooxygenase reaction mechanism of PGHS--evidence for a reversible transition between a pentadienyl radical and a new tyrosyl radical by nitric oxide trapping.

Incubation of prostaglandin H synthase-1 (PGHS-1) under anaerobic conditions with peroxide and arachidonic acid leads to two major radical species: a pentadienyl radical and a radical with a narrow EPR spectrum. The proportions of the two radicals are sensitive to temperature, favoring the narrow radical species at 22 °C. The EPR characteristics of this latter radical are somewhat similar to the previously reported narrow-singlet tyrosine radical NS1a and are insensitive to deuterium labeling of AA. To probe the origin and structure of this radical, we combined EPR analysis with nitric oxide (NO) trapping of tyrosine and substrate derived radicals for both PGHS-1 and -2. Formation of 3-nitrotyrosine in the proteins was analyzed by immunoblotting, whereas NO adducts to AA and AA metabolites were analyzed by mass spectrometry and by chromatography of (14)C-labeled products. The results indicate that both nitrated tyrosine residues and NO-AA adducts formed upon NO trapping. The NO-AA adduct was predominantly an oxime at C11 of AA with three conjugated double bonds, as indicated by absorption at 275 nm and by mass spectral analysis. This adduct amounted to 10% and 20% of the heme concentration of PGHS-1 and -2, respectively. For PGHS-1, the yield of NO-AA adduct matched the yield of the narrow radical signal obtained in parallel EPR experiments. High frequency EPR characterization of this narrow radical, reported in an accompanying paper, supports assignment to a new tyrosyl radical, NS1c, rather than an AA-based radical. To reconcile the results from EPR and NO-trapping studies, we propose that the NS1c is in equilibrium with an AA pentadienyl radical, and that the latter reacts preferentially with NO.

Genetic variation in prostaglandin E2 synthesis and signaling, prostaglandin dehydrogenase, and the risk of colorectal adenoma.

BACKGROUND: Prostaglandins are important inflammatory mediators; prostaglandin E2 (PGE2) is the predominant prostaglandin in colorectal neoplasia and affects colorectal carcinogenesis. Prostaglandins are metabolites of omega-6 and omega-3 polyunsaturated fatty acids; their biosynthesis is the primary target of nonsteroidal anti-inflammatory drugs (NSAID), which reduce colorectal neoplasia risk. METHODS: We investigated candidate and tagSNPs in PGE2 synthase (PGES), PGE2 receptors (EP2 and EP4), and prostaglandin dehydrogenase (PGDH) in a case-control study of adenomas (n = 483) versus polyp-free controls (n = 582) and examined interactions with NSAID use or fish intake, a source of omega-3 fatty acids. RESULTS: A 30% adenoma risk reduction was observed for EP2 4950G>A (intron 1; OR(GA/AA vs. GG), 0.71; 95% confidence interval, 0.52-0.99). For the candidate polymorphism EP4 Val294Ile, increasing fish intake was associated with increased adenoma risk among those with variant genotypes, but not among those with the Val/Val genotype (P(interaction) = 0.02). An interaction with fish intake was also observed for PGES -664A>T (5' untranslated region; P(interaction) = 0.01). Decreased risk with increasing fish intake was only seen among those with the AT or TT genotypes (OR(>2 t/wk vs. <1 t/wk), 0.56; 95% confidence interval, 0.28-1.13). We also detected interactions between NSAIDs and EP2 9814C>A (intron 1) and PGDH 343C>A (intron 1). However, none of the observed associations was statistically significant after adjustment for multiple testing. We investigated potential gene-gene interactions using the Chatterjee 1 degree of freedom Tukey test and logic regression; neither method detected significant interactions. CONCLUSIONS: These data provide little support for associations between adenoma risk and genetic variability related to PGE(2), yet suggest gene-environment interactions with anti-inflammatory exposures.

Prostaglandin H synthase: resolved and unresolved mechanistic issues.

The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.