RESUMEN
Vitamin K is a vital micronutrient implicated in a variety of human diseases. Warfarin, a vitamin K antagonist, is the most commonly prescribed oral anticoagulant. Patients overdosed on warfarin can be rescued by administering high doses of vitamin K because of the existence of a warfarin-resistant vitamin K reductase. Despite the functional discovery of vitamin K reductase over eight decades ago, its identity remained elusive. Here, we report the identification of warfarin-resistant vitamin K reductase using a genome-wide CRISPR-Cas9 knockout screen with a vitamin K-dependent apoptotic reporter cell line. We find that ferroptosis suppressor protein 1 (FSP1), a ubiquinone oxidoreductase, is the enzyme responsible for vitamin K reduction in a warfarin-resistant manner, consistent with a recent discovery by Mishima et al. FSP1 inhibitor that inhibited ubiquinone reduction and thus triggered cancer cell ferroptosis, displays strong inhibition of vitamin K-dependent carboxylation. Intriguingly, dihydroorotate dehydrogenase, another ubiquinone-associated ferroptosis suppressor protein parallel to the function of FSP1, does not support vitamin K-dependent carboxylation. These findings provide new insights into selectively controlling the physiological and pathological processes involving electron transfers mediated by vitamin K and ubiquinone.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , NAD(P)H Deshidrogenasa (Quinona) , Warfarina , Humanos , Anticoagulantes/farmacología , Sistemas CRISPR-Cas , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Ubiquinona/farmacología , Ubiquinona/metabolismo , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/genética , Vitamina K Epóxido Reductasas/metabolismo , Warfarina/farmacología , Proteínas Reguladoras de la Apoptosis/genéticaRESUMEN
UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained Ë 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu.
Asunto(s)
Dimetilaliltranstransferasa , Vitamina K , Animales , Distrofias Hereditarias de la Córnea , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/metabolismo , Células HEK293 , Humanos , Ratones , Mutación , Vitamina K/genética , Vitamina K/metabolismo , Vitamina K 2/metabolismoRESUMEN
γ-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K-dependent (VKD) proteins involved in a wide variety of physiologic processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype-phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of 3 structure-function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter proteins, and then the carboxylation efficiency of these reporter proteins was evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C terminus plays a critical role in the binding of osteocalcin but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein-protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre-messenger RNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes and clarified why vitamin K administration rectified bleeding disorders but not nonbleeding disorders.
Asunto(s)
Ligasas de Carbono-Carbono/genética , Carboxiliasas/genética , Procesamiento Proteico-Postraduccional/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Ligasas de Carbono-Carbono/química , Carboxiliasas/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Genes Reporteros , Estudios de Asociación Genética , Pleiotropía Genética , Células HEK293 , Trastornos Hemorrágicos/tratamiento farmacológico , Trastornos Hemorrágicos/genética , Humanos , Mutación , Mutación Missense , Osteocalcina/genética , Osteocalcina/metabolismo , Proteína C/genética , Proteína C/metabolismo , Dominios Proteicos , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Vitamina K/fisiología , Vitamina K/uso terapéutico , Proteína Gla de la MatrizRESUMEN
BACKGROUND: Vitamin K antagonists (VKAs), such as warfarin, have remained the cornerstone of oral anticoagulation therapy in the prevention and treatment of thromboembolism for more than half a century. They function by impairing the biosynthesis of vitamin K-dependent (VKD) clotting factors through the inhibition of vitamin K epoxide reductase (VKOR). The challenge of VKAs therapy is their narrow therapeutic index and highly variable dosing requirements, which are partially the result of genetic variations of VKOR. OBJECTIVES: The goal of this study was to search for an improved VKA that is tolerant to the genetic variations of its target enzyme. METHODS: A series of vitamin K derivatives with benzyl and related side-chain substitutions at the 3-position of 1,4-naphthoquinone were synthesized. The role of these compounds in VKD carboxylation was evaluated by mammalian cell-based assays and conventional in vitro activity assays. RESULTS: Our results showed that replacing the phytyl side-chain with a methylene cyclooctatetraene (COT) moiety at the 3-position of vitamin K1 converted it from a substrate to an inhibitor for VKD carboxylation. Strikingly, this COT-vitamin K derivative displayed a similar inhibition potency in warfarin-resistant VKOR mutations whose warfarin resistance varied more than 400-fold. Further characterization of COT-vitamin K for the inhibition of VKD carboxylation suggested that this compound targets multiple enzymes in the vitamin K redox cycle. Importantly, the anticoagulation effect of COT-vitamin K can be rescued with high doses of vitamin K1 . CONCLUSION: We discovered a vitamin K analogue that functions as a VKA and is tolerant to genetic variations in the target enzyme.
Asunto(s)
Anticoagulantes , Vitamina K , Animales , Coagulación Sanguínea , Vitamina K 1 , Vitamina K Epóxido Reductasas/genética , WarfarinaRESUMEN
Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Trastornos Hemorrágicos/inducido químicamente , Ensayos Analíticos de Alto Rendimiento/métodos , Vitamina K/metabolismo , 4-Hidroxicumarinas/efectos adversos , 4-Hidroxicumarinas/aislamiento & purificación , 4-Hidroxicumarinas/farmacología , Animales , Anticoagulantes/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Células Hep G2 , Humanos , Indenos/efectos adversos , Indenos/aislamiento & purificación , Indenos/farmacología , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Uso Fuera de lo Indicado , Vitamina K/efectos adversos , Vitamina K/antagonistas & inhibidores , Vitamina K/aislamiento & purificación , Vitamina K/farmacología , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Vitamina K Epóxido Reductasas/metabolismoRESUMEN
Vitamin K-dependent carboxylation is a post-translational modification essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With the discovery of new vitamin K-dependent proteins, the importance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor's propeptide controls substrate binding and product releasing during carboxylation, and the propeptide of factor IX appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin's hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocalcin cannot direct the carboxylation of the coagulation factor, it is required for the efficient carboxylation of osteocalcin. This suggests that the coagulation factors may have a different mechanism of carboxylation from osteocalcin. Together, results from this study provide insight into efficiently controlling one physiological process, such as coagulation without affecting the other, like bone metabolism.
Asunto(s)
Ligasas de Carbono-Carbono , Vitamina K , Animales , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Factor IX/genética , Humanos , Fenotipo , Procesamiento Proteico-PostraduccionalRESUMEN
Despite the difficulty in administering a safe dose regimen and reports of emerging resistance, warfarin (1) remains the most widely-used oral anticoagulant for the prevention and treatment of thrombosis in humans globally. Systematic substitution of the warfarin phenyl ring with either 1,3,5,7-cyclooctatetraene (COT) (2), cubane (3), cyclohexane (4) or cyclooctane (5) and subsequent evaluation against the target enzyme, vitamin K epoxide reductase (VKOR), facilitated interrogation of both steric and electronic properties of the phenyl pharmacophore. The tolerance of VKOR to further functional group modification (carboxylate 14, PTAD adduct 15) was also investigated. The results demonstrate the importance of both annulene conferred π-interactions and ring size in the activity of warfarin.
Asunto(s)
Anticoagulantes/farmacocinética , Anticoagulantes/uso terapéutico , Warfarina/farmacocinética , Warfarina/uso terapéutico , Anticoagulantes/farmacología , Humanos , Warfarina/farmacologíaRESUMEN
Cubane was recently validated as a phenyl ring (bio)isostere, but highly strained caged carbocyclic systems lack π character, which is often critical for mediating key biological interactions. This electronic property restriction associated with cubane has been addressed herein with cyclooctatetraene (COT), using known pharmaceutical and agrochemical compounds as templates. COT either outperformed or matched cubane in multiple cases suggesting that versatile complementarity exists between the two systems for enhanced bioactive molecule discovery.
RESUMEN
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.
Asunto(s)
Anticoagulantes/farmacología , Inhibidores Enzimáticos/farmacología , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Administración Oral , Anticoagulantes/administración & dosificación , Línea Celular , Resistencia a Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Humanos , Fenindiona/administración & dosificación , Fenindiona/análogos & derivados , Fenindiona/farmacología , Mutación Puntual , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/genética , Vitamina K Epóxido Reductasas/metabolismo , Warfarina/administración & dosificación , Warfarina/farmacologíaRESUMEN
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.
Asunto(s)
Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Warfarina/farmacología , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Cisteína/química , Células HEK293 , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación Missense , Mutación Puntual , Unión Proteica , Conformación Proteica , Tirosina/química , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/deficiencia , Vitamina K Epóxido Reductasas/metabolismoRESUMEN
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.
Asunto(s)
Dominio Catalítico/genética , Membrana Celular/metabolismo , Vitamina K Epóxido Reductasas/metabolismo , Vitamina K/metabolismo , Secuencia de Aminoácidos/genética , Línea Celular , Cisteína/química , Células HEK293 , Humanos , Relación Estructura-Actividad , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Vitamina K Epóxido Reductasas/genética , Warfarina/farmacologíaAsunto(s)
Ligasas de Carbono-Carbono/deficiencia , Mutación del Sistema de Lectura , Trastornos Hemorrágicos/genética , Eliminación de Secuencia , Factores de Coagulación Sanguínea/metabolismo , Sistemas CRISPR-Cas , Ligasas de Carbono-Carbono/genética , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes , Genes Reporteros , Células HEK293 , Trastornos Hemorrágicos/sangre , Humanos , Intrones/genética , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Empalme del ARN/genética , Vitamina K/administración & dosificación , Vitamina K/farmacologíaRESUMEN
Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix γ-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo.
Asunto(s)
Ligasas de Carbono-Carbono/genética , Mutación , Sangrado por Deficiencia de Vitamina K/genética , Anomalías Múltiples/genética , Secuencia de Aminoácidos , Secuencia de Bases , Pruebas de Coagulación Sanguínea , Sistemas CRISPR-Cas , Calcinosis/genética , Proteínas de Unión al Calcio/genética , Enfermedades de los Cartílagos/genética , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/genética , Femenino , Genes Reporteros , Estudios de Asociación Genética , Genotipo , Células HEK293 , Deformidades Congénitas de la Mano/genética , Hemorragia , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Fenotipo , Estenosis de la Válvula Pulmonar/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteína Gla de la MatrizRESUMEN
Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.
Asunto(s)
Dominio Catalítico , Secuencia Conservada , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Disulfuros/química , Células HEK293 , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Vitamina K/análogos & derivados , Vitamina K/metabolismoRESUMEN
Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/enzimología , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Oxigenasas de Función Mixta/química , Modelos Moleculares , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Cristalografía por Rayos X , Citoplasma/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Homología Estructural de Proteína , Synechococcus , Vitamina K Epóxido ReductasasRESUMEN
AIMS: Vitamin K epoxide reductase complex, subunit 1 (VKORC1) is a critical participant in the production of active forms of reduced vitamin K and is required for modification of vitamin K-dependent proteins. Homologues of VKORC1 (VKORH) exist throughout evolution, but in bacteria they appear to function in oxidative protein folding as well as quinone reduction. In the current study we explore two questions: Do VKORHs function in the mammalian vitamin K cycle? Is the pair of loop cysteines-C43 and C51 in human VKORC1-conserved in all VKORC1s, essential for the activity of vitamin K epoxide reduction? RESULTS: We used our recently developed cell-based assay to compare the function of VKORHs to that of human VKORC1 in mammalian cells. We identified for the first time a VKORH (from Mycobacterium tuberculosis [Mt-VKORH]) that can function in the mammalian vitamin K cycle with vitamin K epoxide or vitamin K as substrate. Consistent with our previous in vitro results, the loop cysteines of human VKORC1 are not essential for its activity in vivo. Moreover, the corresponding loop cysteines of Mt-VKORH (C57 and C65), which are essential for its activity in disulfide bond formation during protein folding in Escherichia coli, are not required in the mammalian vitamin K cycle. INNOVATION AND CONCLUSIONS: Our results indicate that VKORC1 in eukaryotes and Mt-VKORH in bacteria, that is, in their respective native environments, employ apparently different mechanisms for electron transfer. However, when Mt-VKORH is in the mammalian cell system, it employs a mechanism similar to that of VKORC1.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Mycobacterium tuberculosis/enzimología , Vitamina K/metabolismo , Células Cultivadas , Cisteína/metabolismo , Células HEK293 , Humanos , Vitamina K Epóxido ReductasasRESUMEN
We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH2) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH2, and (4) the primary function of VKOR is the reduction of KO to vitamin K.
Asunto(s)
Transducción de Señal/fisiología , Vitamina K/metabolismo , Anticoagulantes/farmacología , Línea Celular , Técnicas de Laboratorio Clínico , Células HEK293 , Humanos , Oxigenasas de Función Mixta/metabolismo , Transducción de Señal/efectos de los fármacos , Vitamina K 1/análogos & derivados , Vitamina K 1/metabolismo , Vitamina K Epóxido Reductasas , Warfarina/farmacologíaRESUMEN
Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].
Asunto(s)
Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Vitamina K 1/análogos & derivados , Vitamina K 2/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico/genética , Cisteína/química , Disulfuros/química , Inhibidores Enzimáticos/farmacología , Cinética , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vitamina K 1/metabolismo , Vitamina K Epóxido Reductasas , Warfarina/farmacologíaRESUMEN
Previously we reported that we could increase the fraction of carboxylated factor X by reducing the affinity of the propeptide for its binding site on human gamma glutamyl carboxylase. We attributed this to an increased turnover rate. However, even with the reduced affinity propeptide, when sufficient overproduction of factor X is achieved, there is still a significant fraction of uncarboxylated recombinant factor X. We report here that the factor X of such a cell line was only 52% carboxylated but that the fraction of carboxylated factor X could be increased to 92% by coexpressing the recently identified gene for vitamin K epoxide reductase. Because vitamin K is in excess in both the untransfected and vitamin K epoxide reductase (VKOR)-transfected cells, the simplest explanation for this result is that VKOR catalyzes both the reduction of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. In addition to its mechanistic relevance, this observation has practical implications for overproducing recombinant vitamin K-dependent proteins for therapeutic use.