Direct Expression of Adrenodoxin Reductase in Escherchia coli and the Functional Characterization.

Sagara, Yusuke; Wada, Akira; Takata, Yutaka; Waterman, Michael R.; Sekimizu, Kazuhisa; Horiuchi, Tadao

doi:10.1248/bpb.16.627

Cited by 119 publications

(97 citation statements)

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“…Bovine adrenodoxin was expressed in Escherichia coli DH5␣ cells with the expression plasmid pBA1159 (35,36). Bovine NADPH-adrenodoxin reductase was expressed in E. coli JM109 cells as described (36,37), using a pCWori ϩ expression vector (38).…”

Section: Methodsmentioning

confidence: 99%

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Federspiel

Codreanu

Goyal

et al. 2016

Molecular & Cellular Proteomics

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Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the ␤5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these offtarget modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying

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Section: Methodsmentioning

confidence: 99%

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Federspiel

Codreanu

Goyal

et al. 2016

Molecular & Cellular Proteomics

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“…Human recombinant CYP27A1, mitochondrial redox partners adrenodoxin (Adx) and adrenodoxin reductase (Adr), and microsomal P450 oxidoreductase were expressed and purifi ed as described (18)(19)(20)(21). Bovine eyes were obtained from a local slaughterhouse 3-5 h after animals were sacrifi ced.…”

Section: Methodsmentioning

confidence: 99%

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

Heo

Bederman

Mast

et al. 2011

Journal of Lipid Research

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“…Recombinant Adx, Adx mutants, and AdR were purified as described elsewhere. 30,31 Protein concentrations were calculated using ε 414 = 9.8 (mM cm) −1 for Adx 32 and ε 450 = 11.3 (mM cm) −1 for AdR. 33 …”

Section: Mutagenesis Expression and Enzyme Purificationmentioning

confidence: 99%

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

et al. 2007

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Protein phosphorylation is a common regulator of enzyme activity. Chemical modification of a protein surface, including phosphorylation, could alter the function of biological electron-transfer reactions. However, the sensitivity of intermolecular electron-transfer kinetics to post-translational protein modifications has not been widely investigated. We have therefore combined experimental and computational studies to assess the potential role of phosphorylation in electron-transfer reactions. We investigated the steroid hydroxylating system from bovine adrenal glands, which consists of adrenodoxin (Adx), adrenodoxin reductase (AdR), and a cytochrome P450, CYP11A1. We focused on the phosphorylation of Adx at Thr-71, since this residue is located in the acidic interaction domain of Adx, and a recent study has demonstrated that this residue is phosphorylated by casein kinase 2 (CK2) in vitro. 1 Optical biosensor experiments indicate that the presence of this phosphorylation slightly increases the binding affinity of oxidized Adx with CYP11A1 ox but not AdR ox . This tendency was confirmed by K A values extracted from Adx concentration-dependent stopped-flow experiments that characterize the interaction between AdR red and Adx ox or between Adx red and CYP11A1 ox . In addition, acceleration of the electron-transfer kinetics measured with stopped-flow is seen only for the phosphorylated Adx-CYP11A1 reaction. Biphasic reaction kinetics are observed only when Adx is phosphorylated at Thr-71, and the Brownian dynamics (BD) simulations suggest that this phosphorylation may enhance the formation of a secondary Adx-CYP11A1 binding complex that provides an additional electron-transfer pathway with enhanced coupling.

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Direct Expression of Adrenodoxin Reductase in Escherchia coli and the Functional Characterization.

Cited by 119 publications

References 0 publications

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

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