The role of cytochrome b 5 structural domains in interaction with cytochromes P450

Sergeev, G. V.; Gilep, Andrei A.; Usanov, Sergey A.

doi:10.1134/s0006297914050046

Cited by 23 publications

(12 citation statements)

References 29 publications

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“…The hydroxylation reactions occur throughout a person’s lifetime, and when a person approaches adrenarche, the hydroxylated products can be shunted towards the subsequent step of formation of androgenic precursors by CYP17A1 in addition to the production of glucocorticoids [7]. The presence of membrane bound form of cyt b 5 has been known to selectively and significantly enhance the rate of the lyase reaction [8]. Mutations of CYP17A1 surface residues: R347, R358 and K89 which are known to interact with cyt b 5 , have been shown to cause impairment of lyase activity [9].…”

Section: Introductionmentioning

confidence: 99%

“…Clearly, cyt b 5 has a physiological significance in maintaining normal levels of androgen synthesis. The nature of this role has been long debated [8,12–14], with Auchus and coworkers studying lyase activity in recombinant yeast using apo b 5 which stimulated the lyase reaction, leading these workers to conclude that there is no redox role [15]. A similar report from Akhtar and coworkers mentions unpublished results demonstrating lyase enhancement in a reconstituted system utilizing Mn- b 5 [9].…”

Section: Introductionmentioning

confidence: 99%

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Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

Duggal

Liu

Gregory

et al. 2016

Biochemical and Biophysical Research Communications

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Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene- nucleus, and androgen biosynthesis by a second C-C lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ~ 5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe-S vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

Duggal

Liu

Gregory

et al. 2016

Biochemical and Biophysical Research Communications

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“…These values are comparable to K d of the complexes of various cytochromes P450 with their functional partners (CPR, CYB5A, ADX) [23, 34-37]. It is important to note that, while the difference in the K d values of complex formation is about twofold, TBXAS1 • CYP11B2 and TBXAS1 • CYP2E1 interactions are very different in their kinetic parameters.…”

Section: Resultsmentioning

confidence: 52%

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Свирид¹,

Ershov²,

Yablokov³

et al. 2017

Acta Naturae

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Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H2 producing thromboxane A2, the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H2 to 12-HHT/thromboxane A2 proantagonist) and TBXAS1 (Kd = (4.3 ± 0.4) × 10-7 M). Importantly, the TBXAS1•CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids.

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“…Highly purified preparations (>95% by SDS-PAGE) of human NADPH-dependent adrenodoxin reductase (ADR), mitochondrial cytochromes b 5 (CYB5B) were expressed and purified in the Institute of Bioorganic Chemistry NASB (Minsk, Belarus). Methods of their expression in E. coli cells as His-tagged proteins, their isolation, purification and functional analysis have been described earlier [ 29 , 30 , 31 ]. SMAD4 and FECH were expressed in E. coli as His-tagged proteins and purified to homogeneity by means of metal-affinity chromatography followed by chromatography on hydroxyapatite and dialysis [ 8 ].…”

Section: Methodsmentioning

confidence: 99%

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

Ершов

Veselovsky

Mezentsev

et al. 2020

IJMS

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Isatin (indole-2, 3-dione) is a non-peptide endogenous bioregulator exhibiting a wide spectrum of biological activity, realized in the cell via interactions with numerous isatin-binding proteins, their complexes, and (sub) interactomes. There is increasing evidence that isatin may be involved in the regulation of complex formations by modulating the affinity of the interacting protein partners. Recently, using Surface Plasmon Resonance (SPR) analysis, we have found that isatin in a concentration dependent manner increased interaction between two human mitochondrial proteins, ferrochelatase (FECH), and adrenodoxine reductase (ADR). In this study, we have investigated the affinity-enhancing effect of isatin on the FECH/ADR interaction. The SPR analysis has shown that FECH forms not only homodimers, but also FECH/ADR heterodimers. The affinity-enhancing effect of isatin on the FECH/ADR interaction was highly specific and was not reproduced by structural analogues of isatin. Bioinformatic analysis performed using three dimensional (3D) models of the interacting proteins and in silico molecular docking revealed the most probable mechanism involving FECH/isatin/ADR ternary complex formation. In this complex, isatin is targeted to the interface of interacting FECH and ADR monomers, forming hydrogen bonds with both FECH and ADR. This is a new regulatory mechanism by which isatin can modulate protein–protein interactions (PPI).

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The role of cytochrome b 5 structural domains in interaction with cytochromes P450

Cited by 23 publications

References 29 publications

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

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