scholarly journals Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

2022 ◽  
Author(s):  
Thomas Chetot ◽  
Etienne Benoit ◽  
Véronique Lambert ◽  
Virginie Lattard
Keyword(s):  
Vitamin K ◽  
Protein Disulfide Isomerase ◽  
Reductase Activity ◽  
Functional Characterization ◽  
Hek293 Cells ◽  
Vitamin K Epoxide Reductase ◽  
Disulfide Isomerase ◽  
Vitamin K Cycle ◽  
Epoxide Reductase ◽  
Protein Disulfide

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

scholarly journals Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents

1994 ◽  
Vol 297 (2) ◽  
pp. 277-280 ◽  
Author(s):  
H H W Thijssen ◽  
Y P G Janssen ◽  
L T M Vervoort
Keyword(s):  
Vitamin K ◽  
Lipoic Acid ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Vitamin K Cycle ◽  
Redox Active ◽  
Starting Point ◽  
Epoxide Reductase ◽  
Triton X ◽  
Reducing Equivalents

This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.

Effect of warfarin on plasma and liver vitamin K levels and vitamin K epoxide reductase activity in relation to plasma clotting factor levels in rats

1990 ◽  
Vol 57 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Yoshitaka Yamanaka ◽  
Masahiro Yamano ◽  
Kojiro Yasunaga ◽  
Tsutomu Shike ◽  
Kiyohisa Uchida
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Clotting Factor ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase ◽  
Liver Vitamin

Vitamin K epoxide reductase activity in the metabolism of epoxides

1985 ◽  
Vol 34 (15) ◽  
pp. 2617-2620 ◽  
Author(s):  
I. Liptay-Reuter ◽  
K. Dose ◽  
T. Guenthner ◽  
W. Wörner ◽  
F. Oesch
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase

scholarly journals A cysteine-based redox switch regulates millisecond dynamics of protein disulfide isomerase and controls enzyme activity

2020 ◽  
Author(s):  
Mathivanan Chinnaraj ◽  
Robert Flaumenhaft ◽  
Nicola Pozzi
Keyword(s):  
Single Molecule ◽  
Protein Disulfide Isomerase ◽  
Equilibrium Distribution ◽  
Reductase Activity ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disulfide Isomerase ◽  
Redox Switch ◽  
Protein Disulfide

AbstractCysteine residues provide enzymes involved in signaling and metabolism with an allosteric mechanism to quickly respond to changes in the surrounding redox environment. Yet how thiol modifications impact enzyme structure and dynamics is poorly understood. Here, we apply single-molecule Förster resonance energy transfer (smFRET) to study redox-dependent conformational dynamics of a highly flexible oxidoreductase, protein disulfide isomerase (PDI), in solution. We demonstrate that PDI toggles in the millisecond timescale between two major conformational ensembles, “open” and “closed”, that differ ∼20Å in length due to relocation of the two catalytic domains, whose equilibrium distribution is modulated by the redox state. While reduced PDI predominantly populates the open ensemble, oxidized PDI visits both ensembles with similar probability. We provide evidence that i) transition from the open to the closed ensemble requires loss of free thiols, not disulfide bonding, as previously thought, and ii) exposure to small molecules that alkylate the catalytic cysteines and mutation of cysteines in the N-terminal active site shift PDI to the closed conformation. Finally, using mutational and kinetic analyses, we show that the fraction of open ensemble at equilibrium positively correlates with the reductase activity of PDI. This work forms the basis of a new dynamic mechanism of PDI regulation by a cysteine-based redox switch in which thiol-controlled equilibrium distribution of the open/closed ensembles dictates function.

Purification of a Single Peptide with Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH2 Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 331-331
Author(s):  
Pei-hsuan Chu ◽  
Teng-yi Huang ◽  
Jason Williams ◽  
Darrel W. Stafford
Keyword(s):  
Vitamin K ◽  
Complete Recovery ◽  
Vitamin K Epoxide Reductase ◽  
Vitamin K Cycle ◽  
The Us ◽  
Epoxide Reductase ◽  
Measurable Activity ◽  
Turn Over ◽  
Single Peptide

Abstract More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

scholarly journals Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway ofAspergillus niger

2000 ◽  
Vol 66 (2) ◽  
pp. 775-782 ◽  
Author(s):  
Celina Ngiam ◽  
David J. Jeenes ◽  
Peter J. Punt ◽  
Cees A. M. J. J. Van Den Hondel ◽  
David B. Archer
Keyword(s):  
Protein Disulfide Isomerase ◽  
Secretory Pathway ◽  
Functional Characterization ◽  
Killer Toxin ◽  
Rnase A ◽  
Secretory Proteins ◽  
Mrna Levels ◽  
Western Blots ◽  
Disulfide Isomerase ◽  
Protein Disulfide

ABSTRACT Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene,pdiA, encoding PDIA was previously isolated fromAspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A.pdiA also complemented PDI function in aSaccharomyces cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy

1989 ◽  
Vol 38 (16) ◽  
pp. 2693-2701 ◽  
Author(s):  
Takashi Matsubara ◽  
Akira Touchi ◽  
Toshio Harauchi ◽  
Kyoji Takano ◽  
Toshio Yoshizaki
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase
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