Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

doi:10.1016/j.bbrc.2005.08.129

Biochemical and Biophysical Research Communications

Volume 336, Issue 2, 21 October 2005, Pages 565-571

https://doi.org/10.1016/j.bbrc.2005.08.129 Get rights and content

Abstract

Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions.

Section snippets

Materials and methods

Cell culture and cell lines. HEK 7X cell lines were obtained from the American Tissue Culture Collection and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) with 5% FBS and in DMEM containing 800 μg/ml geniticin (G418, Life Technologies) as a selection drug used for the stable line. Stable and transient cell lines were used. Cells were transfected with FLAG-tagged NR1 and 6-His-tagged DCS-1 constructs in pCEP4 vector [19] using the LipofectAMINE reagent (Life

Cooperative action of NR1 and DCS-1

In this study, we used menadione as a model quinone and examined its cytotoxicity in stable HEK cell lines over-expressing NR1. The cytotoxicity of menadione was compared between HEK cells stably expressing NR1 transfected with: (1) the β-galactosidase reporter gene or (2) DCS-1. Additionally, HEK cells expressing endogenous levels of NR1 were transfected with β-galactosidase as a control. Our results show that menadione induced cell death in cells expressing NR1, as indicated by an increased

Discussion

We have isolated a novel human NADPH-dependent flavin reductase, NR1, from a variety of human tissues including kidney, placenta, and brain. This is a human ortholog of C. elegans fre-1[19]. Previous study shows that NR1 is also expressed in various cancer cell lines [17]. NR1 belongs to a family of flavin-containing proteins and shows strong sequence conservation in the regions shown to be involved in FMN, FAD, and NADPH cofactor binding. The N-terminus of human NADPH-cytochrome P450 reductase

Acknowledgment

We thank Vincent Marra for technical assistance.

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Notably, human DcpS binds NR1 and can significantly reduce the menadione-induced cytotoxicity. After exposure to menadione, cells expressing both NR1 and DcpS are differentiated and healthy, whereas cells expressing NR1 alone are rounded and shrivelled [29]. This clearly indicates DcpS importance in human physiology and health.
Decapping Scavenger (DcpS) enzyme rids eukaryotic cells of short mRNA fragments containing the 5′ mRNA cap structure, which appear in the 3′ → 5′ mRNA decay pathway, following deadenylation and exosome-mediated turnover. The unique structural properties of the cap, which consists of 7-methylguanosine attached to the first transcribed nucleoside by a triphosphate chain (m⁷GpppN), guarantee its resistance to non-specific exonucleases. DcpS enzymes are dimers belonging to the Histidine Triad (HIT) superfamily of pyrophosphatases. The specific hydrolysis of m⁷GpppN by DcpS yields m⁷GMP and NDP. By precluding inhibition of other cap-binding proteins by short m⁷GpppN-containing mRNA fragments, DcpS plays an important role in the cap-dependent mRNA metabolism. Over the past decade, lots of new structural, biochemical and biophysical data on DcpS has accumulated. We attempt to integrate these results, referring to DcpS enzymes from different species. Such a synergistic characteristic of the DcpS structure and activity might be useful for better understanding of the DcpS catalytic mechanism, its regulatory role in gene expression, as well as for designing DcpS inhibitors of potential therapeutic application, e.g. in spinal muscular atrophy.
The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway
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Moreover, we have identified and characterised a gene family consisting of three putative CPRs in T. cruzi named TcCPR-A, TcCPR-B and TcCPR-C (Portal et al., 2008). Interestingly, phylogenetic tree analysis revealed that TcCPR-A and other related CPRs clustered with human NR1 (Portal et al., 2008), a cytoplasmic flavoprotein oxidoreductase which appears widely expressed in human cancer cell lines (Paine et al., 2000; Kwasnicka-Crawford and Vincent, 2005). A common feature of all CPRs belonging to this cluster is the absence of the characteristic N-terminal transmembrane domain.
Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP⁺/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.
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Cytochrome P450 hemoproteins (CYPs) are involved in the synthesis of endogenous compounds such as steroids, fatty acids and prostaglandins as well as in the activation and detoxification of foreign compounds including therapeutic drugs. Cytochrome P450 reductase (CPR, E.C.1.6.2.4) transfers electrons from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. This work presents the complete sequences of three non-allelic CPR genes from Trypanosoma cruzi. The encoded proteins named TcCPR-A, TcCPR-B and TcCPR-C have calculated molecular masses of 68.6 kDa, 78.4 kDa and 71.3 kDa, respectively. Deduced amino acid sequences share 11% amino acid identity, possess the conserved binding domains for FMN, FAD and NADPH and differ in the hydrophobic 27-amino acid residues of the N-terminal extension, which is absent in TcCPR-A. Every T. cruzi CPRs, TcCPR-A, TcCPR-B and TcCPR-C, were cloned and expressed in Escherichia coli. All of the recombinant enzymes reduced cytochrome c in a NADPH absolutely dependent manner with low K_m values for this cofactor. They all were also strongly inhibited by diphenyleneiodonium, a classical flavoenzyme inhibitor. In addition, TcCPRs could support CYP activities when assayed in reconstituted systems containing rat liver microsomes. Polyclonal antiserum rose against the recombinant enzymes TcCPR-A and TcCPR-B demonstrated its presence in every T. cruzi developmental stages, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole. We suggest a participation of TcCPR-B in the detoxification metabolism of the parasite.
Restricted role for methionine synthase reductase defined by subcellular localization
2008, Molecular Genetics and Metabolism
Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase, J. Biol. Chem. 279 (2004) 47536–47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.
Regulation of mRNA decapping
2010, Wiley Interdisciplinary Reviews: RNA

^☆: This work was supported by the Canadian Institutes of Health Research and the Canadian Stroke Network.

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Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity☆

Abstract

Section snippets

Materials and methods

Cooperative action of NR1 and DCS-1

Discussion

Acknowledgment

Biochim. Biophys. Acta

Pharmacol. Ther.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Eur. J. Pharmacol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Chem. Biol. Interact.

Pharmacol. Ther.

J. Biol. Chem.

J. Biol. Chem.

Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains

Proc. Natl. Acad. Sci. USA

Expression of mammalian cytochromes P450 in yeast

Methods Mol. Biol.