NQO1 in Protection Against Oxidative Stress

NQO1 in protection against oxidative stress

NAD(P)H:quinone acceptor oxidoreductases  (NQO’s),  as their  name suggests, are efficient at reducing endogenous and exogenous quinones to hydroquinones [1]. The enzymes are present across species including an ancient NQO3 subfamily in eubacteria and most probably evolved as defense systems against environmental quinones [2]. In mammalian systems, there are two major forms of NQO’s; NQO1 and NQO2. This brief perspective will focus on NQO1 and its role in protection against stress and in particular, oxidative stress.

With respect to the mechanism of enzymatic reduction, NQO1 is a flavoprotein and the FAD contained in the enzyme is key to both its stability and function. The apo or FAD-deficient form of NQO1 is degraded rapidly by the proteasome [3;4]. NQO1*2, a highly prevalent polymorphic form, of NQO1 does not bind FAD efficiently and is also degraded rapidly by the proteasome [5-7]. NQO1 is somewhat unusual in that it can utilize both NADH and NADPH as cofactors to reduce the flavin and that reduction occurs via a hydride transfer mechanism so that NQO1 functions, at least with respect to quinone reduction, as a two electron reductase resulting in direct generation of the hydroquinone species [8-10]. The second mammalian form of NQO, NQO2, lacks an intact pyridine nucleotide binding site at the C-terminal of the protein and as a result functions more effectively with reduced nicotinamide riboside (NRH) as a cofactor.

Quinones are electrophilic molecules and many are capable of alkylating proteins and DNA in cells. Quinones can also be reduced via one electron reductases to produce semiquinone radicals and reactive oxygen radicals. As a result, two electron reduction of quinones to hydroquinones by NQO1 is often considered as a detoxification step. A good example would be the case of metabolism of the quinone 2-methyl-1,4-naphthoquinone or menadione in liver systems where increased NQO1 leads to diversion of the quinone from one electron reductases leading to decreased superoxide and singlet oxygen formation and decreased toxicity [11;12]. However, if reduction of a quinone by NQO1 generates a hydroquinone that is unstable to oxygen, generation of oxidative stress may occur after autoxidation of the hydroquinone [13;14]. The redox stability of the hydroquinone is therefore critical in classification of this reaction as a detoxification or toxification mechanism. Examples of the production of redox unstable hydroquinones by NQO1 include β-lapachone [15] and streptonigrin [16].

Induction of NQO1 under stress conditions

One of the notable characteristics of NQO1 is that it is highly inducible by multiple forms of stress. The first report on DT-diaphorase (NQO1) was published in 1958 [17] although the enzyme was likely the same  as a vitamin K reductase isolated earlier by Martius [18] .  Studies on enzyme induction occurred soon after isolation of the enzyme. Treatment of rats and mice with low doses of polycyclic aromatic hydrocarbons (PAHs) increased NQO1 activity and also protected against toxicity and carcinogenicity [19]  This was essentially the start of many studies on the relationship of induction of NQO1 to chemoprotection. Talalay and coworkers validated and extended this  hypothesis primarily using PAHs, azo dyes and phenolic antioxidants [20-23]. Subsequent work has shown that there is a broad diversity in the types of compound that can induce NQO1 including hydrogen peroxide [24]. Induction of NQO1 was also observed after exposure  to ionizing radiation  [25], photodynamic therapy [26] , nanoparticle exposure [27] and shear stress in blood vessels. [28]. Early studies of induction of NQO1 showed up to 10 fold induction using aromatic hydrocarbons [21] and up to 30 fold induction at the mRNA level by X ray radiation in cells [25]. The great diversity of NQO1 inducers can be explained in large part by considerations of the mechanisms underlying enzyme induction. Considerable work led to identification of the critical XRE and ARE promoter elements in the NQO1 gene that controlled  induction as a coordinated response to cellular stress [29-33]. This led to the conclusion that NQO1 can be induced via Nrf2-mediated induction  and similarly via Ah receptor-mediated induction  [30;31;34-36] so  a wide range of stimuli can lead to elevated levels of NQO1.  Tumor cells which are continually under a variety of stresses often express high levels of NQO1 [37] arguably as a means to adapt to the multiple stresses caused by metabolic dysregulation. Interestingly, both NQO1 and Nrf2 have been shown to drive tumor growth in certain cases [38;39]. The high levels of NQO1 in solid tumors has been used as a therapeutic target for the design of antitumor agents that can be activated by the enzyme [40].

Why induce NQO1 under stress conditions?

The rapid induction of NQO1 by various stressors raises the question as to what beneficial effects NQO1 might have for a cell under stress. It is unlikely that the ability of the enzyme to reduce exogenous quinones is the predominant reason for its induction except in very specific cases. NQO1 however is a pleiotropic enzyme [41;42] and its multiple functions both in the realm of catalytic reduction of endogenous substrates and its ability to bind other macromolecules such as proteins and RNA potentially influencing their function may provide clues to answering this question.  Under conditions of oxidative stress, consideration of some of the known endogenous substrates for NQO1 may provide plausible mechanisms as to why NQO1 might be induced and the resultant beneficial effects at the cellular level.

Potential endogenous substrates for NQO1

A) CoQ derivatives. Beyer and colleagues showed that NQO1 could reduce CoQ derivatives of various chain lengths including CoQ₉ and CoQ₁₀ to generate effective antioxidant forms of these molecules [43]. For this mechanism to function effectively in cells, NQO1-catalyzed reduction would need to occur in membranes or at membrane interfaces where the longer chain forms of CoQ exert their antioxidant protection. NQO1 was found to be capable of maintaining the reduced form of CoQ₉ and CoQ₁₀ in unilamellar or multilamellar vesicles and protecting membrane components including mitochondrial membranes of hepatocytes from free radical damage and lipid peroxidation [43;44].

B) Vitamin E quinone (-tocopherol quinone, TQ) is formed from vitamin E (-tocopherol) after free radical attack in membrane systems but unlike Vitamin E, has no inherent antioxidant activity. NQO1-mediated reduction of TQ generates the hydroquinone derivative (TQ-HQ) of TQ, which has potent antioxidant activity, and cells transfected with increasing levels of NQO1 generated higher levels of TQ-HQ and were better protected against cumene hydroperoxide-induced lipid peroxidation [45].

Reduced forms of both Vitamin E and CoQ₁₀ are important components of the plasma membrane redox system which serves to protect cells against oxidative stress and lipid peroxidation  [46]. The generation of antioxidant forms of CoQ₁₀ and Vitamin E may be an important physiological function of NQO1. The suggestion has been made that NQO1 was selected during evolution as a CoQ reductase and its conversion of xenobiotics and other synthetic molecules was secondary and coincidental to its primary effects on ubiquinone [43].

C) Superoxide

NQO1 exerts limited activity as a superoxide reductase which has been demonstrated directly using EPR and in cell free and cellular studies [45]. The ability of NQO1 to reduce superoxide is dependent upon generation of FADH₂ (flavinhydroquinone) following enzyme mediated hydride transfer from reduced pyridine nucleotide cofactors to FAD. These results are consistent with previous studies by Massy et al.  which describe similar reactions with enzyme-free flavinhydroquinone and superoxide [9]. The flavin in NQO1 is not covalently bound so the ability to reduce superoxide to hydrogen peroxide is likely a function of the FAD content of the enzyme. The rate constant of this reaction is less than an order of magnitude greater than spontaneous dismutation of superoxide [45] so the enzymatic reaction is unlikely to be relevant unless NQO1 is expressed at very high levels or other flavoproteins contribute to this effect. Siegel et al. demonstrated the cellular relevance by overexpressing NQO1 at high levels which resulted in increased superoxide scavenging [45]. Basal NQO1 levels in some cells are very high and, as stated previously, induction of NQO1 to high levels may also occur under various stresses. In cardiac cells including human vascular smooth muscle and endothelial cells for example, NQO1 levels are relatively high whereas SOD content is relatively low. NQO1 in cardiac cells was found to be capable of scavenging NQO1 which was blocked by NQO1 inhibition and increased by NQO1 induction [47]. Whether superoxide reacts with other flavoproteins with similar redox chemistry to NQO1 or whether these reactions are specific to NQO1 is unknown. There are many flavin-containing enzymes and the flavoproteome contains approximately 90 proteins, the vast majority of which are involved in redox reactions [48]. It is interesting to speculate that if other flavoproteins are inducible by oxidative stress and capable of similar flavin redox chemistry as NQO1 they may contribute further to superoxide reductase activity. Interestingly, FAD containing proteins such as thioredoxin reductase and glutathione reductase are components of the Nrf2 inducible gene battery in mammalian systems [49] both of which can be switched on by oxidative stress.

NQO1 as an intracellular source of NAD⁺

Turnover of NQO1 in the presence of a reducing co-substrate results in the conversion of NADH to NAD⁺ which can be utilized for fueling the activity of poly ADP ribose polymerase (PARP) and sirtuins. Oxidative stress may result in damage to DNA at multiple levels and adequate PARP activity contributes to DNA repair although extensive PARP activation can lead to cell death [50].  Sirtuins have been shown to play a role in protection against oxidative stress [51] and SIRT 3 for example, inhibits mitochondrial reactive oxygen production [52]. Hepatic-specific knockout of SIRT1 resulted in augmented markers of liver oxidative stress relative to control mice [53] and SIRT1 has also been shown to protect against oxidative stress in the heart [54].

Macromolecular binding functions of NQO1

The complexity linked to the molecular functions of NQO1 has increased considerably over the last decade with the demonstration that NQO1 stabilizes certain proteins against degradation. The ability of NQO1 to bind to other proteins is now well-characterized and these interactions have relevance for cellular protection. Many of the proteins reported to be stabilized by NQO1 have downstream functional or transcriptional effects so the effects of NQO1 at the level of a single protein may well be amplified. An excellent example is the stabilization of the tumor suppressor gene p53. NQO1 associates with p53 in a protein-protein interaction protecting it against ubiquitin-independent 20S proteasomal degradation [55] NQO1 has been shown to stabilize a growing number of proteins including p53, p63 p73, ornithine decarboxylase and PGC-1 [56-59]. NQO1 was recently shown to stabilize HIF-1 in RKO colon tumors in mice xenografts as a result of impaired proteasomal degradation leading to increased tumor growth  [38]. An interesting hypothesis is that many of the proteins whose stability is regulated by NQO1 via the 20S proteasome are intrinsically disordered proteins [60;61]. 26S proteasome mediated degradation is ubiquitin-dependent and requires recognition of the polyubiquitinated molecule, deubiquitination and unfolding and translocation of the protein into the 20S proteasome particle where degradation takes place. Degradation of proteins by the 20S proteasome on the other hand is ubiquitin-independent and relies on the inherent structural disorder of the protein being degraded [60]. The mechanism of stabilization by NQO1 of proteins normally subjected to 20S proteasomal degradation was proposed to occur through binding of NQO1 to the specific protein in question and this has indeed been shown to be the case with many of the proteins stabilized by NQO1. However other potential NQO1-mediated mechanisms of regulation at the level of the 20S proteasome have been explored. Moscovitz et al., [61] demonstrated in a cell free system that NQO1 binds directly to the purified 20S proteasome. A double negative feedback loop between the 20S proteasome and NQO1 was suggested where binding of NQO1 prevented the proteolytic activity of the 20S proteasome while the 20S proteasome degraded the apo form of NQO1 which, due to its lack of FAD, remains partially unfolded [61]. These data suggest a link between FAD content which reflects nutritional status and the protein degradation and quality control systems of the cell [62]. Specifically at the level of NQO1, Moscovitz et al.  [61] proposed that as cells become more metabolically active and produce more oxidative stress, cellular FAD will increase leading to greater NQO1 stability and oxidative stress protection. Direct binding of NQO1 to the proteasome is also supported by data in yeast where an ortholog of NQO1 binds to the 20S proteasome  [63]. It should be emphasized that the precise mechanisms whereby NQO1 stabilizes proteins either at the level of interaction with proteins undergoing degradation and/or direct association with the 20S proteasome remain to be fully characterized. However, collectively these findings suggest that one rationale for inducing NQO1 under stress conditions is to control degradation of proteins important to cellular survival.

RNA binding

RNA – protein interactome studies in mammalian cells unexpectedly identified metabolic enzymes including NQO1 as RNA binding proteins [64] and reinforced earlier findings of binding between metabolic enzymes and RNA in yeast [65;66].  Of 23 such protein families identified, 13 of them bound  ATP, UTP, GTP  or pyridine nucleotides suggesting that protein domains commonly involved in nucleotide binding such as the Rossman fold may represent RNA-binding interfaces [64;67]. Specifically with respect to NQO1, DiFrancesco et al. [68] recently demonstrated binding of NQO1 to a subset of mRNA’s in HepG2 cells  including SERPIN1A1 which codes for alpha 1 antitrypsin resulting in increased translation. The ability of NQO1 to interact with RNA is novel and demonstrates the potential complexity of the effect of NQO1 on multiple regulatory systems. It also raises the possibility that NQO1 may exert effects not only at the level of protein degradation by the 20S proteasome but also affect translation as a result of interaction with mRNA.

A redox sensitive molecular switch in macromolecular binding ?

It is intriguing that the ability of NQO1 to bind to other proteins [55] and RNA [68] has been found to depend on the pyridine nucleotide redox balance. Binding of NAD(P)H to NQO1 has been proposed to change the conformational structure of NQO1 to allow oxidized NAD(P)⁺ to be ejected and subsequent binding of quinone [10;69]. Chen et al. [70] demonstrated using purified rat NQO1 that at least one of the biochemical consequences of this conformational change was that after binding of NADPH the exposed C-terminal regions of the protein were protected against proteolytic digestion. Our own work, (Siegel and Ross, unpublished) shows that the NQO1 structure is modified under different NADH/NAD⁺ ratios and these structural changes modulate the binding of NQO1 to antibodies impacting their ability to immunoprecipitate the protein. Under conditions of oxidative stress, it is conceivable that altered pyridine nucleotide ratios could serve as a signal resulting in change of NQO1 structure and binding to a different set of proteins and RNA under oxidative conditions.

Conclusion

NQO1 can be induced by multiple forms of stress including oxidative stress which may not be surprising given the multiple roles of NQO1 in the cell.  NQO1 can contribute to protection by provision of antioxidant forms of physiological substrates including ubiquinone and vitamin E quinone and provision of NAD⁺ for sirtuins and PARP. The direct scavenging of superoxide by NQO1 may also provide protection against oxidative stress particularly where cellular SOD levels are low. The macromolecular binding functions of NQO1 may be just as relevant to a cellular protective response. Binding of NQO1 to proteins, particularly intrinsically disordered proteins, can stabilize them against 20S proteasomal degradation while binding of NQO1 to mRNA can increase translation of proteins. This suggests that one important role for NQO1 when induced under oxidative stress conditions is to maintain the stability of critical proteins. Changes in pyridine nucleotide redox ratios modulate the conformation and binding of NQO1 to other proteins and mRNA. Whether redox specific binding of NQO1 to other proteins and mRNA are relevant mechanisms of protection or signaling in oxidative stress remains to be elucidated.

Reference List

1.  Ernster L: DT-diaphorase. Meth.Enzymol. 1967, 10:309-317.

2.  Vasiliou V, Ross D, Nebert DW: Update of the NAD(P)H:quinone oxidoreductase (NQO) gene family. Hum.Genomics 2006, 2:329-335.

3.  Lienhart WD, Gudipati V, Uhl MK, Binter A, Pulido SA, Saf R, Zangger K, Gruber K, Macheroux P: Collapse of the native structure caused by a single amino acid exchange in human NAD(P)H:quinone oxidoreductase(1.). FEBS J. 2014, 281:4691-4704.

4.  Pey AL, Megarity CF, Timson DJ: FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1. Biochim.Biophys.Acta 2014, 1842:2163-2173.

5.  Siegel D, Anwar A, Winski SL, Kepa JK, Zolman KL, Ross D: Rapid Polyubiquitination and Proteasomal Degradation of a Mutant Form of NAD(P)H:Quinone Oxidoreductase 1. Mol.Pharmacol. 2001, 59:263-268.

6.  Traver RD, Horikoshi T, Danenberg KD, Stadlbauer TH, Danenberg PV, Ross D, Gibson NW: NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. Cancer Res. 1992, 52:797-802.

7.  Traver RD, Siegel D, Beall HD, Phillips RM, Gibson NW, Franklin WA, Ross D: Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase). Br.J.Cancer 1997, 75:69-75.

8.  Hosoda S, Nakamura W, Hayashi K: Properties and reaction mechanism of DT diaphorase from rat liver. J.Biol.Chem. 1974, 549:6416-6423.

9.  Tedeschi G, Chen S, Massey V: DT-diaphorase.  Redox potential, steady-state, and rapid reaction studies. J.Biol.Chem. 1995, 270:1198-1204.

10.  Bianchet MA, Faig M, Amzel LM: Structure and mechanism of NADPH:quinone acceptor oxidoreductases (NQO). Meth.Enzymol. 2004, 382:144-174.

11.  Lind C, Hochstein P, Ernster L: DT-Diaphorase as a quinone reductase: A cellular control device against semiquinone and superoxide radical formation. Arch.Biochem.Biophys. 1982, 216:178-185.

12.  Thor H, Smith MT, Hartzell P, Bellomo G, Jewell SA, Orrenius S: The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implication of oxidative stress in intact cells. J.Biol.Chem. 1982, 257:12419-12425.

13.  Cadenas E, Hochstein P, Ernster L: Pro-and antioxidant functions of quinones and quinone reductases in mammalian cells. Adv.Enzymol.Relat.Areas Mol.Biol. 1992, 65:97-146.

14.  Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, Siegel D: NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem.Biol.Interact. 2000, 129:77-97.

15.  Pink JJ, Planchon SM, Tagliarino C, Varnes ME, Siegel D, Boothman DA: NAD(P)H:Quinone Oxidoreductase Activity Is the Principal Determinant of beta-Lapachone Cytotoxicity. J.Biol.Chem. 2000, 275:5416-5424.

16.  Beall HD, Liu Y, Siegel D, Bolton EM, Gibson NW, Ross D: Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. Biochem.Pharmacol. 1996, 51:645-652.

17.  Ernster L, Navazio F: Soluble diaphorase in animal tissues. Acta Chem.Scand. 1958, 12:595-602.

18.  Martius C: Die stellung des phyllochinons (Vitamin K1) in der atmungskette. Biochem.Z. 1954, 326:26-27.

19.  Huggins C, Fukunishi R: Induced protection of adrenal cortex against 7,12- dimethylbenz(a)anthracene. Influence of ethionine. Induction of menadione reductase. Incorporation of thymidine-H3. J.Exp.Med. 1964, 119:923-942.

20.  Benson AM, Hunkler MJ, Talalay P: Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc.Natl.Acad.Sci.USA 1980, 77:5216-5220.

21.  De Long MJ, Prochaska HJ, Talalay P: Induction of NAD(P)H:quinone reductase in murine hepatoma cells by phenolic antioxidants, azo dyes and other chemoprotectors: A model system for he study of anticarcinogens. Proc.Natl.Acad.Sci.USA 1986, 83:787-791.

22.  Talalay P, Prochaska HJ: Mechanisms of induction of NAD(P)H:quinone reductase. Chemica Scripta 1987, 27A:61-66.

23.  Prochaska HJ, Santamaria AB, Talalay P: Rapid detection of inducers of enzymes that protect against carcinogens. Proc.Natl.Acad.Sci.USA 1992, 89:2394-2398.

24.  Favreau LV, Pickett CB: Transcriptional regulation of the rat NAD(P)H: quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-o-tertradecanoylphorbal ester, 13-acetate. J.Biol.Chem. 1993, 268:19875-19881.

25.  Boothman D, Meyers M, Fukunaga N, Lee SW: Isolation of x ray-inducible transcripts from radioresistant human melanoma cells. Proc.Natl.Acad.Sci.USA 1993, 90:7200-7204.

26.  Lamberti MJ, Vittar NB, da Silva FC, Ferreira VF, Rivarola VA: Synergistic enhancement of antitumor effect of beta-Lapachone by photodynamic induction of quinone oxidoreductase (NQO1). Phytomedicine. 2013, 20:1007-1012.

27.  Zhang H, Liu H, Davies KJ, Sioutas C, Finch CE, Morgan TE, Forman HJ: Nrf2-regulated phase II enzymes are induced by chronic ambient nanoparticle exposure in young mice with age-related impairments. Free Radic.Biol.Med. 2012, 52:2038-2046.

28.  Chen XL, Varner SE, Rao AS, Grey JY, Thomas S, Cook CK, Wasserman MA, Medford RM, Jaiswal AK, Kunsch C: Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism. J.Biol.Chem. 2003, 278:703-711.

29.  Jaiswal AK, McBride OW, Adesnik M, Nebert DW: Human dioxin-inducible cytosolic NAD(P)H:menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16. J.Biol.Chem. 1988, 263:13572-13578.

30.  Nebert DW, Petersen DD, Fornace Jr. AJ: Cellular responses to oxidative stress: The [Ah] gene battery as a paradigm. Environ.Health Perspect. 1990, 88:13-25.

31.  Favreau LV, Pickett CB: Transcriptional regulation of the rat NAD(P)H:quinone oxidoreductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J.Biol.Chem. 1991, 266:4556-4551.

32.  Jaiswal AK: Antioxidant response element. Biochem.Pharmacol. 1994, 48:439-444.

33.  Nioi P, McMahon M, Itoh K, Yamamoto M, Hayes JD: Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence. Biochem.J. 2003, 374:337-348.

34.  Jaiswal AK: Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic.Biol.Med 2004, 36:1199-1207.

35.  Nioi P, Hayes JD: Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors. Mutat.Res. 2004, 555:149-171.

36.  Nebert DW, Puga A, Vasiliou V: Role of the Ah receptor and the dioxin-inducible [Ah] gene battery in toxicity, cancer, and signal transduction. Ann.N.Y.Acad.Sci. 1993, 685:624-640.

37.  Siegel D, Franklin WA, Ross D. Immunohistochemical detection of NAD(P)H:quinone oxidoreductase in human lung and lung tumors. Clinical Cancer Research 4, 3083-3088. 1998.

Ref Type: Generic

38.  Oh ET, Kim JW, Kim JM, Kim SJ, Lee JS, Hong SS, Goodwin J, Ruthenborg RJ, Jung MG, Lee HJ, Lee CH, Park ES, Kim C, Park HJ: NQO1 inhibits proteasome-mediated degradation of HIF-1alpha. Nat.Commun. 2016, 7:13593.

39.  DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, Mangal D, Yu KH, Yeo CJ, Calhoun ES, Scrimieri F, Winter JM, Hruban RH, Iacobuzio-Donahue C, Kern SE, Blair IA, Tuveson DA: Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011, 475:106-109.

40.  Siegel D, Yan C, Ross D: NAD(P)H:quinone oxidoreductase 1 (NQO1) in the sensitivity and resistance to antitumor quinones. Biochem.Pharmacol. 2012, 83:1033-1040.

41.  Ross D: Quinone reductases.Multitasking in the metabolic world. Drug Metabolism Reviews 2004, 36:639-654.

42.  Dinkova-Kostova AT, Talalay P: NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector. Arch.Biochem.Biophys. 2010.

43.  Beyer RE, Segura-Aguilar J, Di Bernardo S, Cavazzoni M, Fato R, Fiorentini D, Galli M, Setti M, Landi L, Lenaz G: The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc.Natl.Acad.Sci.USA 1996, 93:2528-2532.

44.  Landi L, Fiorentini D, Galli MC, Segura-Aguilar J, Beyer RE: DT-diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function. Free Radic.Biol.Med. 1997, 22:329-335.

45.  Siegel D, Gustafson DL, Dehn DL, Han JY, Boonchoong P, Berliner LJ, Ross D: NAD(P)H:quinone oxidoreductase 1: Role as a superoxide scavenger. Molecular Pharmacology 2004, 65:1-10.

46.  Hyun DH, Hernandez JO, Mattson MP, de CR: The plasma membrane redox system in aging. Ageing Res.Rev. 2006, 5:209-220.

47.  Zhu H, Jia Z, Mahaney JE, Ross D, Misra HP, Trush MA, Li Y: The highly expressed and inducible endogenous NAD(P)H:quinone oxidoreductase 1 in cardiovascular cells acts as a potential superoxide scavenger. Cardiovasc.Toxicol. 2007, 7:202-211.

48.  Lienhart WD, Gudipati V, Macheroux P: The human flavoproteome. Arch.Biochem.Biophys. 2013, 535:150-162.

49.  Hayes JD, Dinkova-Kostova AT: The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem.Sci. 2014, 39:199-218.

50.  Bai P: Biology of Poly(ADP-Ribose) Polymerases: The Factotums of Cell Maintenance. Mol.Cell 2015, 58:947-958.

51.  Osborne B, Bentley NL, Montgomery MK, Turner N: The role of mitochondrial sirtuins in health and disease. Free Radic.Biol.Med. 2016, 100:164-174.

52.  Bell EL, Emerling BM, Ricoult SJ, Guarente L: SirT3 suppresses hypoxia inducible factor 1alpha and tumor growth by inhibiting mitochondrial ROS production. Oncogene 2011, 30:2986-2996.

53.  Yang Y, Zhang S, Fan C, Yi W, Jiang S, Di S, Ma Z, Hu W, Deng C, Lv J, Li T, Nie Y, Jin Z: Protective role of silent information regulator 1 against hepatic ischemia: effects on oxidative stress injury, inflammatory response, and MAPKs. Expert.Opin.Ther.Targets. 2016, 20:519-531.

54.  Hsu CP, Odewale I, Alcendor RR, Sadoshima J: Sirt1 protects the heart from aging and stress. Biol.Chem 2008, 389:221-231.

55.  Asher G, Lotem J, Kama R, Sachs L, Shaul Y: NQO1 stabilizes p53 through a distinct pathway. Proc.Natl.Acad.Sci.U.S.A 2002, 99:3099-3104.

56.  Asher G, Lotem J, Cohen B, Sachs L, Shaul Y: Regulation of p53 stability and p53-dependent apoptosis by NADH quinone oxidoreductase 1. Proc.Natl Acad.Sci.U.S.A 2001, 98:1188-1193.

57.  Asher G, Bercovich Z, Tsvetkov P, Shaul Y, Kahana C: 20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1. Mol.Cell 2005, 17:645-655.

58.  Adamovich Y, Shlomai A, Tsvetkov P, Umansky KB, Reuven N, Estall JL, Spiegelman BM, Shaul Y: The protein level of PGC-1alpha, a key metabolic regulator, is controlled by NADH-NQO1. Mol.Cell Biol. 2013, 33:2603-2613.

59.  Hershkovitz RO, Shpilberg O, Granot G: NAD(P)H quinone oxidoreductase protects TAp63gamma from proteasomal degradation and regulates TAp63gamma-dependent growth arrest. PLoS.ONE. 2010, 5:e11401.

60.  Ben-Nissan G, Sharon M: Regulating the 20S proteasome ubiquitin-independent degradation pathway. Biomolecules. 2014, 4:862-884.

61.  Moscovitz O, Tsvetkov P, Hazan N, Michaelevski I, Keisar H, Ben-Nissan G, Shaul Y, Sharon M: A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1. Mol.Cell 2012, 47:76-86.

62.  Martinez-Limon A, Alriquet M, Lang WH, Calloni G, Wittig I, Vabulas RM: Recognition of enzymes lacking bound cofactor by protein quality control. Proc.Natl.Acad.Sci.U.S.A 2016, 113:12156-12161.

63.  Sollner S, Schober M, Wagner A, Prem A, Lorkova L, Palfey BA, Groll M, Macheroux P: Quinone reductase acts as a redox switch of the 20S yeast proteasome. EMBO Rep. 2009, 10:65-70.

64.  Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann BM, Strein C, Davey NE, Humphreys DT, Preiss T, Steinmetz LM, Krijgsveld J, Hentze MW: Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 2012, 149:1393-1406.

65.  Scherrer T, Mittal N, Janga SC, Gerber AP: A screen for RNA-binding proteins in yeast indicates dual functions for many enzymes. PLoS.ONE. 2010, 5:e15499.

66.  Tsvetanova NG, Klass DM, Salzman J, Brown PO: Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS.ONE. 2010, 5.

67.  Castello A, Hentze MW, Preiss T: Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins. Trends Endocrinol.Metab 2015, 26:746-757.

68.  Di FA, Di GC, Panda AC, Huynh P, Peaden R, Navas-Enamorado I, Bastian P, Lehrmann E, Diaz-Ruiz A, Ross D, Siegel D, Martindale JL, Bernier M, Gorospe M, Abdelmohsen K, de CR: Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation. Free Radic.Biol.Med. 2016, 99:225-233.

69.  Faig M, Bianchet MA, Talalay P, Chen S, Winski S, Ross D, Mario AL: Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: Species comparison and structural changes with substrate binding and release. Proc.Natl.Acad.Sci.U.S.A 2000, 97:3177-3182.

70.  Chen S, Deng PS, Bailey JM, Swiderek KM: A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase. Protein Sci. 1994, 3:51-57.