Search Results (6)

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme that converts vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents supplied by its redox partner through thiol–disulphide exchange reactions. The functionally related molecular complexes assembled during this process have never been described, except for a proposed de novo model of a ‘precursor’ complex of hVKORC1 associated with protein disulphide isomerase (PDI). Using numerical approaches (in silico modelling and molecular dynamics simulation), we generated alternative 3D models for each molecular complex bonded either covalently or non-covalently. These models differ in the orientation of the PDI relative to hVKORC1 and in the cysteine residue involved in forming protein–protein disulphide bonds. Based on a comparative analysis of these models’ shape, folding, and conformational dynamics, the most probable putative complexes, mimicking the ‘precursor’, ‘intermediate’, and ‘successor’ states, were suggested. In addition, we propose using these complexes to develop the ‘allo-network drugs’ necessary for treating blood diseases. Full article

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme transforming vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents delivered by its redox partner through thiol-disulfide exchange reactions. The luminal loop (L-loop) is the principal mediator of hVKORC1 activation, and it is a region frequently harbouring numerous missense mutations. Four L-loop hVKORC1 mutants, suggested in vitro as either resistant (A41S, H68Y) or completely inactive (S52W, W59R), were studied in the oxidised state by numerical approaches (in silico). The DYNASOME and POCKETOME of each mutant were characterised and compared to the native protein, recently described as a modular protein composed of the structurally stable transmembrane domain (TMD) and the intrinsically disordered L-loop, exhibiting quasi-independent dynamics. The DYNASOME of mutants revealed that L-loop missense point mutations impact not only its folding and dynamics, but also those of the TMD, highlighting a strong mutation-specific interdependence between these domains. Another consequence of the mutation-induced effects manifests in the global changes (geometric, topological, and probabilistic) of the newly detected cryptic pockets and the alternation of the recognition properties of the L-loop with its redox protein. Based on our results, we postulate that (i) intra-protein allosteric regulation and (ii) the inherent allosteric regulation and cryptic pockets of each mutant depend on its DYNASOME; and (iii) the recognition of the redox protein by hVKORC1 (INTERACTOME) depend on their DYNASOME. This multifaceted description of proteins produces “omics” data sets, crucial for understanding the physiological processes of proteins and the pathologies caused by alteration of the protein properties at various “omics” levels. Additionally, such characterisation opens novel perspectives for the development of “allo-network drugs” essential for the treatment of blood disorders. Full article

Human vitamin K epoxide reductase (hVKORC1) enzymatic activity requires an initial activation by a specific redox protein, a less studied step in the hVKORC1 vital cycle. Significant steric conditions must be met by enzymes, being that to adapt their configurations is mandatory for hVKORC1 activation. We studied, by molecular dynamics (MD) simulations, the folding and conformational plasticity of hVKORC1 in its inactive (fully oxidised) state using available structures, crystallographic and from de novo modelling. According to the obtained results, hVKORC1 is a modular protein composed of the stable transmembrane domain (TMD) and intrinsically disordered luminal (L) loop, possessing the great plasticity/adaptability required to perform various steps of the activation process. The docking (HADDOCK) of Protein Disulfide Isomerase (PDI) onto different hVKORC1 conformations clearly indicated that the most interpretable solutions were found on the target closed L-loop form, a prevalent conformation of hVKORC1’s oxidised state. We also suggest that the cleaved L-loop is an appropriate entity to study hVKORC1 recognition/activation by its redox protein. Additionally, the application of hVKORC1 (membrane protein) in aqueous solution is likely to prove to be very useful in practice in either in silico studies or in vitro experiments. Full article

Background: This study aimed to investigate the effects of genetic variants and haplotypes in the renin–angiotensin system (RAS) on the risk of warfarin-induced bleeding complications at therapeutic international normalized ratios (INRs). Methods: Four single nucleotide polymorphisms (SNPs) of AGT, two SNPs of REN, three SNPs of ACE, four SNPs of AGTR1, and one SNP of AGTR2, in addition to VKORC1 and CYP2C9 variants, were investigated. We utilized logistic regression and several machine learning methods for bleeding prediction. Results: The study included 142 patients, among whom 21 experienced bleeding complications. We identified a haplotype, H2 (TCG), carrying three SNPs of ACE (rs1800764, rs4341, and rs4353), which showed a significant relation with bleeding complications. After adjusting covariates, patients with H2/H2 experienced a 0.12-fold (95% CI 0.02–0.99) higher risk of bleeding complications than the others. In addition, G allele carriers of AGT rs5050 and A allele carriers of AGTR1 rs2640543 had 5.0- (95% CI 1.8–14.1) and 3.2-fold (95% CI 1.1–8.9) increased risk of bleeding complications compared with the TT genotype and GG genotype carriers, respectively. The AUROC values (mean, 95% CI) across 10 random iterations using five-fold cross-validated multivariate logistic regression, elastic net, random forest, support vector machine (SVM)–linear kernel, and SVM–radial kernel models were 0.732 (0.694–0.771), 0.741 (0.612–0.870), 0.723 (0.589–0.857), 0.673 (0.517–0.828), and 0.680 (0.528–0.832), respectively. The highest quartile group (≥75th percentile) of weighted risk score had approximately 12.0 times (95% CI 3.1–46.7) increased risk of bleeding, compared to the 25–75th percentile group, respectively. Conclusion: This study demonstrated that RAS-related polymorphisms, including the H2 haplotype of the ACE gene, could affect bleeding complications during warfarin treatment for patients with mechanical heart valves. Our results could be used to develop individually tailored intervention strategies to prevent warfarin-induced bleeding. Full article

Redox (reduction–oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol–disulphide exchange reactions between PDI and hVKORC1. Full article

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 (vkorc1) and vkorc1 like 1 (vkorc1l1) were determined during the larval ontogeny of Senegalese sole (Solea senegalensis), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1, and ovary and brain for ssvkorc1l1. Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions. Full article