Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents.
Ontology highlight
ABSTRACT:
SUBMITTER: Thijssen HH
PROVIDER: S-EPMC1137825 | biostudies-other | 1994 Jan
REPOSITORIES: biostudies-other
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Project description:Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in ?-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be ?-carboxylated, and mapped the ?-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is ?-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPAR?) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPAR? and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.
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Project description:Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain gamma-carboxylation of many blood coagulation factors. Here, we report the 3.6 A crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.
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Project description:Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the gamma-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-alpha, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-alpha interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.
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Project description:Vitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K-dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarin's inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarin's binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKOR's endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarin's mechanism of anticoagulation.
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Project description:Warfarin, acenocoumarol, phenprocoumon, and fluindione are commonly prescribed oral anticoagulants for the prevention and treatment of thromboembolic disorders. These anticoagulants function by impairing the biosynthesis of active vitamin K-dependent coagulation factors through the inhibition of vitamin K epoxide reductase (VKOR). Genetic variations in VKOR have been closely associated with the resistant phenotype of oral anticoagulation therapy. However, the relative efficacy of these anticoagulants, their mechanisms of action, and their resistance variations among naturally occurring VKOR mutations remain elusive. Here, we explored these questions using our recently established cell-based VKOR activity assay with the endogenous VKOR function ablated. Our results show that the efficacy of these anticoagulants on VKOR inactivation, from most to least, is: acenocoumarol > phenprocoumon > warfarin > fluindione. This is consistent with their effective clinical dosages for stable anticoagulation control. Cell-based functional studies of how each of the 27 naturally occurring VKOR mutations responds to these 4 oral anticoagulants indicate that phenprocoumon has the largest resistance variation (up to 199-fold), whereas the resistance of acenocoumarol varies the least (<14-fold). Cell-based kinetics studies show that fluindione appears to be a competitive inhibitor of VKOR, whereas warfarin is likely to be a mixed-type inhibitor of VKOR. The anticoagulation effect of these oral anticoagulants can be reversed by the administration of a high dose of vitamin K, apparently due to the existence of a different enzyme that can directly reduce vitamin K. These findings provide new insights into the selection of oral anticoagulants, their effective dosage management, and their mechanisms of anticoagulation.
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Project description:Vitamin K epoxide reductase (VKOR) is an essential enzyme for vitamin K-dependent carboxylation, while the physiological function of its paralogous enzyme VKOR-like (VKORL) is yet unknown. Although these two enzymes share approximately 50% protein sequence homology, the membrane topology of VKOR is still in debate. Here, we explored the differences in the membrane topology and disulfide-linked oligomerization of these two enzymes. Results from mutating the critical amino acid residues in the disputed transmembrane (TM) regions revealed that the second TM domain in the proposed 4-TM model of VKOR does not function as an authentic TM helix; supporting VKOR is a 3-TM protein, which is different from VKORL. Additionally, altering the loop sequence between the two conserved cysteine residues of VKORL affects its activity, supporting the notion that the conserved loop cysteines of VKORL are involved in its active site regeneration. However, a similar mutation in VKOR does not affect its enzymatic activity. Finally, our results show that although both VKOR and VKORL form disulfide-linked oligomers, the cysteine residues involved in the oligomerization appear to be different. Overall, the structural and functional differences between VKOR and VKORL shown here indicate that VKORL might have a different physiological function other than recycling vitamin K.
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Project description:Intracellular thiols like L-cysteine and glutathione play a critical role in the regulation of cellular processes. Escherichia coli has multiple L-cysteine transporters, which export L-cysteine from the cytoplasm into the periplasm. However, the role of L-cysteine in the periplasm remains unknown. Here we show that an L-cysteine transporter, YdeD, is required for the tolerance of E. coli cells to hydrogen peroxide. We also present evidence that L-cystine, a product from the oxidation of L-cysteine by hydrogen peroxide, is imported back into the cytoplasm in a manner dependent on FliY, the periplasmic L-cystine-binding protein. Remarkably, this protein, which is involved in the recycling of the oxidized L-cysteine, is also found to be important for the hydrogen peroxide resistance of this organism. Furthermore, our analysis of the transcription of relevant genes revealed that the transcription of genes encoding FliY and YdeD is highly induced by hydrogen peroxide rather than by L-cysteine. These findings led us to propose that the inducible L-cysteine/L-cystine shuttle system plays an important role in oxidative stress tolerance through providing a reducing equivalent to the periplasm in E. coli.
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Project description:Microsomal epoxide hydrolase/epoxide hydrolase 1 (mEH/EPHX1) works in conjunction with cytochromes P450 to metabolize a variety of compounds, including xenobiotics, pharmaceuticals and endogenous lipids. mEH has been most widely studied for its role in metabolism of xenobiotic and pharmaceutical compounds where it converts hydrophobic and reactive epoxides to hydrophilic diols that are more readily excreted. Inhibition or genetic disruption of mEH can be deleterious in the face of many industrial, environmental or pharmaceutical exposures and EPHX1 polymorphisms are associated with the development of exposure-related cancers. The role of mEH in endogenous epoxy-fatty acid (EpFA) metabolism has been less well studied. In vitro, mEH metabolizes most EpFAs at a far slower rate than soluble epoxide hydrolase (sEH) and has thus been generally considered to exert a minor role in EpFA metabolism in vivo. Indeed, sEH inhibitors or sEH-deficiency increase EpFA levels and are protective in animal models of cardiovascular disease. Recently, however, mEH was found to have a previously unrecognized and substantial role in EpFA metabolism in vivo. While few studies have examined the role of mEH in cardiovascular homeostasis, there is now substantial evidence that mEH can regulate cardiovascular function through regulation of EpFA metabolism. The discovery of a prominent role for mEH in epoxyeicosatrienoic acid (EET) metabolism, in particular, suggests that additional studies on the role of mEH in cardiovascular biology are warranted.
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Project description:Polymorphisms in the vitamin K-epoxide reductase complex subunit 1 gene, Vkorc1, could affect blood coagulation and other vitamin K-dependent proteins, such as osteocalcin (bone Gla protein, BGP). Here we sequenced the Vkorc1 gene in 40 mouse priority strains. We analyzed Vkorc1 haplotypes with respect to prothrombin time (PT) and bone mineral density and composition (BMD and BMC); phenotypes expected to be vitamin K-dependent and represented by data in the Mouse Phenome Database (MPD).In the commonly used laboratory strains of Mus musculus domesticus we identified only four haplotypes differing in the intron or 5' region sequence of the Vkorc1. Six haplotypes differing by coding and non-coding polymorphisms were identified in the other subspecies of Mus. We detected no significant association of Vkorc1 haplotypes with PT, BMD and BMC within each subspecies of Mus. Vkorc1 haplotype sequences divergence between subspecies was associated with PT, BMD and BMC.Phenotypic variation in PT, BMD and BMC within subspecies of Mus, while substantial, appears to be dominated by genetic variation in genes other than the Vkorc1. This was particularly evident for M. m. domesticus, where a single haplotype was observed in conjunction with virtually the entire range of PT, BMD and BMC values of all 5 subspecies of Mus included in this study. Differences in these phenotypes between subspecies also should not be attributed to Vkorc1 variants, but should be viewed as a result of genome wide genetic divergence.
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Project description:Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we conducted structure simulations, which revealed a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.
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