MINIREVIEW New roles of flavoproteins in molecular cell biology: An unexpected role for quinone reductases as regulators of proteasomal degradation Sonja Sollner and Peter Macheroux Technische Universita Graz, Institut fur Biochemie, Austria ăt ă Keywords avin; NAD(P)H; ornithine decarboxylase; oxidative stress; peptide flip; proteasome; redox state; reduction; transcription factors; ubiquitination Correspondence P Macheroux, Graz University of Technology, Institute of Biochemistry, Petersgasse 12 ⁄ II, A-8010 Graz, Austria Fax: +43 316 873 6952 Tel: +43 316 873 6450 E-mail: peter.macheroux@tugraz.at (Received December 2008, revised 29 April 2009, accepted May 2009) doi:10.1111/j.1742-4658.2009.07143.x Quinone reductases are ubiquitous soluble enzymes found in bacteria, fungi, plants and animals These enzymes utilize a reduced nicotinamide such as NADH or NADPH to reduce the flavin cofactor (either FMN or FAD), which then affords two-electron reduction of cellular quinones Although the chemical nature of the quinone substrate is still a matter of debate, the reaction appears to play a pivotal role in quinone detoxification by preventing the generation of potentially harmful semiquinones In recent years, an additional role of quinone reductases as regulators of proteasomal degradation of transcription factors and possibly intrinsically unstructured protein has emerged To fulfil this role, quinone reductase binds to the core particle of the proteasome and recruits certain transcription factors such as p53 and p73a to the complex The latter process appears to be governed by the redox state of the flavin cofactor of the quinone reductase, thus linking the stability of transcription factors to cellular events such as oxidative stress Here, we review the current evidence for protein complex formation between quinone reductase and the 20S proteasome in eukaryotic cells and describe the regulatory role of this complex in stabilizing transcription factors by acting as inhibitors of their proteasomal degradation Introduction Quinones are abundant cyclic organic compounds present in the environment as well as in pro- and eukaryotic cells They can be reduced by two- or oneelectron reduction to either the hydroquinone or the semiquinone form A number of organisms express enzymes that afford strict two-electron reduction to the hydroquinone form in an attempt to avoid the generation of semiquinones This species is known to cause oxidative stress by reacting with molecular oxygen, eventually leading to the generation of superoxide radicals (redox cycling) Hence, quinone reductases (QRs) from pro- and eukaryotes have a protective effect against quinone-related oxidative cell damage Consequently, QRs have been identified in bacteria, fungi, plants and mammals Originally, QRs were classified as ‘DT-diaphorases’ to express the fact that they utilize both DNPH (reduced diphosphopyridine nucleotide, NADH) and TPNH (reduced triphosphopyridine nucleotide, NADPH) as a source of reducing equivalents [1] At the time, the term ‘diaphorase’ was used to describe an enzyme (preferentially a flavoprotein) capable of transferring electrons from reduced pyrimidine nucleotides to electron acceptors [2] This nomenclature led to confusion because ‘diaphorase activity’ could be detected in numerous biological systems The first ‘DT-diapho- Abbreviations NQO, mammalian NAD(P)H:quinone oxidoreductase; ODC, ornithine decarboxylase; QR, quinone reductase; ROS, reactive oxygen species FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4313 Quinone reductase as regulator of the proteasome S Sollner and P Macheroux rase’, reported by Ernster & Navazio [3], is now known as mammalian NAD(P)H:quinone oxidoreductase (NQO1, isozyme 1) However, the acronym NQO has traditionally been confined to QRs from mammalian sources Although the first successful crystallization of QR was reported in the late 1980s [4,5], it was another couple of years before Li and coworkers eventually solved the structure of rat liver NAD(P)H:quinone oxidoreductase [6] The crystal structure revealed that the fold of the N-terminal portion resembles that of flavodoxin, a bacterial electron-transfer protein involved in a variety of photosynthetic and nonphotosynthetic reactions [7] The biological unit for NQO1, as for most QRs studied to date, is a dimer The overall fold of the flavodoxin-like catalytic domain consists of a twisted, central five-stranded parallel b sheet surrounded by helices The FAD cofactor is noncovalently bound at the interface of the monomers, with the redox-active isoalloxazine ring positioned at one side of two equivalent crevices, thereby forming two identical, independent active sites [6] Structure determination of NQO2 confirmed the close structural relationship between NQO1 and NQO2 Similar to NQO1, NQO2 self-associates as a homodimer and contains two identical catalytic sites located at opposite ends of the dimer interface Each catalytic site forms a large cavity, lined by residues from both peptide chains with the flavin isoalloxazine ring forming the bottom [8] Interestingly, the flavodoxin-like structure of QRs is not restricted to mammalian enzymes, but is conserved down to yeast Lot6p, a homologous QR from the unicellular model organism Saccharomyces cerevisiae, also adopts a dimeric flavodoxin-like fold but binds one FMN cofactor per protomer instead of FAD [9] The unique property of QRs is their ability to transfer two electrons to a quinone, thereby catalyzing the formation of a two-electron reduced hydroquinone without the generation of a one-electron reduced semiquinone [10] This feature seems to be crucial for understanding the physiological role of QRs as a cellular device to avoid the formation of semiquinones and hence the generation of harmful reactive oxygen species (ROS) Although some QRs can utilize both NADH and NADPH as a source of electrons (e.g NQO1, Lot6p), others have developed a strong preference for either NADH (e.g AzoA from Enterococcus faecalis [11]) or NADPH (e.g YhdA from Bacillus subtilis [12]) By contrast, NQO2 is unable to employ NADH or NADPH as a source of electrons, but instead uses reduced N-ribosyl- and N-alkyl-dihydronicotinamide 4314 However, the issue of oxidizing substrates seems to be far more complicated It is generally assumed that enzymes involved in the detoxification mechanisms of xenobiotics not possess endobiotic substrates but have evolved in such a way that a broad range of chemical structures can be processed In fact, the size and shape of the catalytic sites of NQO1, NQO2 and Lot6p suggest that these enzymes have evolved to accept a variety of ring-containing substrates Nevertheless, a number of naturally occurring quinones comprising vitamin K derivatives (menaquinone and phylloquinone), coenzyme Q (ubiquinone) and dopaquinone have also been shown to be substrates for mammalian QRs [13] The functional importance of QRs has been a matter of discussion since their discovery The discovery that a vitamin K reductase, described by Maerki & Martius [14], and the DT-diaphorase first described by Ernster & Navazio [3] is actually the same enzyme added to speculation revolving around the physiological role of QRs The opinion that mammalian QRs are primarily involved in xenobiotic metabolism and in preventing the carcinogenicity and toxicity of highly reactive compounds is a more recent development An explicit mechanism by which NQO1 might protect cells was first provided by Iyanagi & Yamazaki [10] by distinguishing between flavoproteins that catalyze oneelectron reductions and those, like NQO1, that catalyze strict two-electron reductions Accordingly, two-electron reduction of quinones can avert: (a) oneelectron redox cycling, which generates highly ROS; and (b) depletion of cellular glutathione by decreasing the levels of quinones, which react easily with thiol groups Furthermore, in contrast to semiquinone products generated by one-electron reducing flavoproteins, the hydroquinone products of the NQO1 pathway are not only more stable, but can also be further metabolized to glucuronide and sulfate conjugation products, thereby facilitating their excretion Thus, the possibility of forming reactive semiquinone radicals, potential mediators of oxidative stress, is highly reduced [15], and consequently, QRs have been proposed to serve as a cellular control device against quinone toxicity A new role for an old enzyme: QR and the proteasome Many proteins have a dual biological role and participate in regulatory cellular functions in addition to their metabolic function as an enzyme [16] For example, glutathione S-transferase associates with c-Jun N-terminal kinase leading to inhibition of kinase acti- FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS S Sollner and P Macheroux Quinone reductase as regulator of the proteasome vity and modulation of signalling and cellular proliferation [17] Similarly, recent studies have demonstrated that eukaryotic QRs bind to the 20S proteasome and affect the lifetime of several transcription factors and ornithine decarboxylase by inhibiting their ubitquitindependent and -independent proteasomal degradation This role of QRs is the focus of this minireview Before we shed further light on this recently discovered function of a historically old enzyme family, we provide a brief introduction to the role of the proteasome A more detailed description of the structure and function of the proteasome is given in several recent review articles [18–20] The bulk of cellular proteins in eukaryotic cells are degraded by the 26S proteasome This 2.5 MDa proteolytic machinery consists of a 20S barrel-structured core that provides a catalytic chamber and a 19S regulatory particle This latter protein complex binds to the edges of the core particle and regulates access to the catalytic chamber The process that leads to proteasomal degradation is initiated by selective polyubiquitination followed by recognition of the condemned protein through the 19S regulatory caps Ubiquitin consists of 76 amino acids and is covalently attached in a highly regulated multistep process to the substrate protein [21–23] The 19S caps are involved in recognition of the polyubiquitinated protein substrates, unfolding of the condemned protein [24], removing ubiquitin chains for recycling [25,26] and opening an axial gate into the 20S catalytic chamber [27] Whereas 26S proteasomal degradation requires ubiquitination of substrate proteins, the 20S proteasome degrades structurally abnormal, misfolded or highly oxidized proteins in a ubiquitin-independent manner under conditions of cellular stress [28] In mammalian cells (cytoplasm and nucleus), most of the 20S core particles are present in their free uncapped form with only a smaller fraction being capped with the 19S regulatory protein complex [29] The association of mammalian QR with the 20S proteasome was first described by Shaul and coworkers in the course of their investigation into the degradation of transcription factors They found that the vast majority of QR (NQO1) from mouse liver extract is bound to the 20S, but not the 26S, proteasome [30] Similar results were obtained with extracts from human red blood cells and with different commercially available proteasome preparations [31] This link between NQO1 and the 20S proteasome at the protein level is also reflected at the transcriptional level: Nrf2, a transcription factor that is activated upon oxidative stress, is a major transcriptional activator of NQO1 [32] Furthermore, activation of the Nrf2 pathway by treatment with 3H-1,2-dithiole-3thione also leads to enhanced expression of most of the 20S proteasome subunits (a1, a2, a4–a7, b1–b6) [33] The demonstration that a complex between QR and the 20S proteasome exists in mammalian cells prompted further experiments in the unicellular model organism S cerevisiae (baker’s yeast) It could be shown that Lot6p, a QR and ortholog of human NQO1, is physically associated with the 20S proteasome [34] Using the 20S proteasome and recombinant Lot6p, several biochemical issues revolving around the stoichiometry and importance of the flavin cofactor could be resolved Fluorescence titration studies exploiting the intrinsic fluorescence of the flavin cofactor demonstrated that two QR dimers bind to one 20S proteasome core particle (Fig 1) [34] Furthermore, QR lacking both flavin cofactors (apoLot6p) was unable to bind to the proteasome, indicating that the presence of the flavin cofactor is required for complex formation Not surprisingly, the enzymatic activity of the QR is also compromised in the presence of proteasome, supporting the direct or indirect involvement of the flavin cofactor in protein complex formation [34] Mdm2 dependent polyubiquitination Fig Proposed mechanisms of ubiquitindependent (right) and ubiquitin-independent (left) degradation pathways of p53 Free p53 (red) binds to the preformed 20S proteasome–QR (green and pale green, respectively) complex after reduction of the QR After reoxidation of the QR flavin cofactor, p53 is released from the complex, becomes ubiquitinated (blue) and is eventually degraded by the 26S proteasome NQO1 (reduced) p53 19 S cap p53 α α β β β p53 Stabilization Degradation α 20S proteasome FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS β α 26S proteasome 19S cap Ubiquitin 4315 Quinone reductase as regulator of the proteasome S Sollner and P Macheroux The impact of the 20S proteasome on QR activity raised the question of whether a reciprocal effect on proteasomal activity occurs or, in other words, does the QR act as a gatekeeper for the proteasome, as recently suggested [35]? Detailed analysis of the trypsin-like, chymotrypsin-like and peptidyl-glutamylprotein-hydrolysing or caspase-like activity [36] demonstrated up to 10 times lower proteolytic activity in the presence of Lot6p [34] Because substrate access through the gated entry port in the outer a-rings of the core particle is considered to be the rate-limiting step in catalysis, it appears likely that the QR binds to or near the a-rings of the proteasome, thereby affecting access to the catalytic chamber, leading to reduced proteasomal activity [37] In this context, it is important to emphasize that it is not at all clear whether this effect on proteasomal activity is part of a regulatory mechanism because 20S proteasome core particles outnumber QR molecules by a factor of 10 [38] Thus, it appears that the majority of 20S core particles exist in their ‘free’ form and only some are associated with Lot6p However, if the number of QR molecules increases in the cells, for example, by overexpression during oxidative stress, it is conceivable that 20S proteasomal activity is severely downregulated by binding of QR Contradictory results concerning the physical association of the mammalian NQO1 and 20S proteasome were recently reported by Jaiswal’s group: although copurification of the 20S proteasome and QR from mouse liver cytosol was observed, they were unable to detect complex formation by immunoprecipitation using a 20S proteasome antibody Therefore, these authors conclude that mammalian QR and the 20S proteasome not form a protein complex, as proposed by others [39] Unfortunately, no explanation for the copurification of QR and the 20S proteasome is provided in this report and the failure to detect the protein complex by immunoprecipitation was not confirmed by an independent method Hence, the relevance of their findings concerning binding of mammalian QR to the 20S proteasome remains unclear at present Protection of transcription factors from proteasomal degradation through association with the QR–proteasome complex Protein degradation is a key cellular process involved in almost every aspect of the living cell [21,22] The prevailing concept assumes that proteins are not proteolysed unless marked by polyubiquitination, a 4316 prerequisite for recognition and degradation by the 26S proteasome [31] For example, the transcription factor and tumour suppressor p53 is subject to ubiquitin-dependent proteasomal degradation [41,42] and only accumulates following various types of stress, leading to growth arrest and apoptosis Ubiquitination of p53 is carried out by Mdm2, an E3 ubiquitin ligase, which binds to the N-terminus of the transcription factor Stabilization of p53 towards proteolysis can be achieved by disruption of the p53– Mdm2 interaction, thereby preventing Mdm2 from ubiquitinating p53 The main process that leads to disruption of the Mdm2 recognition of p53 involves post-translational modifications Accumulation of p53 then results in the expression of a variety of genes necessary to cope with DNA damage and other forms of cellular stress [43] The ubiquitin-dependent degradation of p53 and other proteins appears to be the main pathway for regulating proteolysis by the 26S proteasome The discovery by Shaul and coworkers that p53 (and other proteins as well) is degraded more rapidly when human QR (NQO1) is inhibited by dicoumarol, a potent and specific inhibitor of QRs, was the first hint that another and different regulatory pathway may exist in eukaryotic cells As a result of enhanced degradation of p53 and hence lower levels of the transcription factor, p53-dependent apoptosis in both c-irradiated normal thymocytes and in myeloid leukaemic cells was suppressed These effects could be prevented by overexpression of NQO1, supporting the idea that it might be involved in regulating cellular p53 levels [44] These findings raised questions concerning the role of NQO1 in ubiquitin-dependent proteasomal degradation Does NQO1 affect the ubiquitination process directly or is it involved in an alternative pathway? Further studies addressing this question revealed that regulation of p53 degradation by NQO1 does not require ubiquitination by Mdm2 Instead, a variant of p53, which is resistant to Mdm2-mediated degradation, was shown to be susceptible to dicoumarol-induced degradation, indicating that NQO1-regulated proteasomal p53 degradation is Mdm2 independent Accordingly, two alternative pathways for p53 proteasomal degradation have been proposed: one is ubiquitin dependent and regulated by Mdm2, whereas the other is ubiquitin independent and regulated by the QR NQO1, implying that p53 stabilization is not solely dependent on inhibition of the p53–Mdm2 interaction but also requires physical association with NQO1 (Fig 1) [45] Accumulation of p53 also leads to expression of the PIG3 (QR homologue) and FDXR (ferredoxin reduc- FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS S Sollner and P Macheroux tase) genes and stabilizes p66Shc (the 66 kDa isoform of ShcA, an adaptor protein that relays extracellular signals downstream of receptor tyrosine kinases) The latter protein gives rise to increased levels of intracellular ROS, thereby promoting apoptosis of damaged cells [46,47] This generation of ROS increases expression of NQO1 [48] which then further stabilizes p53 This sequence of events is consistent with the proposed feed-forward loop for p53 stabilization by ROS [46] The association of human QR with transcription factors is not restricted to p53 Following the discovery of p53 as an interaction partner of NQO1, p73 was also reported to be regulated by a ubiquitin-independent process [30] p73, also known as tumour protein 73 (TP73), was the first identified homologue of the tumour suppressor p53 Overexpression, and thus accumulation, of p73 in cultured cells promotes growth arrest and ⁄ or apoptosis similarly to p53 [49,50] The p73 gene encodes a protein with significant sequence and functional similarity to p53 Like p53, p73 has several variants It is expressed as distinct forms differing either at the C- or the N-terminus Currently, six different C-terminal splicing variants have been found in normal cells The a-splice variant of p73 (p73a) contains an additional structural domain near its C-terminus known as the sterile a-motif that is probably responsible for regulating the p53-like functions of p73 [51] This motif appears to be essential for interaction with NQO1 and subsequent stabilization as the p73b isoform lacking the C-terminal sterile a-motif domain was not protected against 20S proteasomal degradation Recently, levels of the tumour suppressor p33ING1b have also been found to be regulated by NQO1 The ING1 gene was originally identified through subtractive hybridization between normal human mammary epithelial cells and seven breast cancer cell lines, and subsequent in vivo selection of genetic suppressor elements that displayed oncogenic characteristics [52] Three alternatively spliced transcripts of the ING1 gene have been found, encoding protein variants with a predicted size of 47, 33 and 24 kDa p33ING1b (ING1b for inhibitor of growth family, member 1b) has been reported to be downregulated in several carcinomas The protein was shown to be a major player in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair [53] Phosphorylation of p33ING1b at Ser126 was reported to be important for proliferation in malinoma cells, as well as modulation of its degradation [54] Garate et al recently detected this tumour suppressor in purified fractions of 20S proteasome and provided evidence that p33ING1b is degraded by the 20S proteasome Quinone reductase as regulator of the proteasome Further results indicate that NQO1 inhibits the degradation of p33ING1b and that ultraviolet irradiation stabilizes p33ING1b by inducing phosphorylation at Ser126, thereby enhancing its interaction with NQO1 [55] Protection of transcription factors through association with a QR is not restricted to NQO1 NQO2, a homologue of NQO1, was shown to prevent transcription factors from being degraded as well [39] Human QR was described for the first time in 1961 as an unknown mammalian cytosolic FAD-dependent flavoprotein catalyzing the oxidation of reduced N-ribosyland N-alkyldihydronicotinamides by menadione and other quinones, but not the oxidation of NADH, NADPH or NMNH (reduced nicotinamide mononucleotide) The enzyme was extensively characterized, but was completely forgotten for several decades In the early 1990s, Jaiswal and coworkers isolated and described a second NAD(P)H:quinone oxidoreductase, which they discovered in the course of cloning human NQO1, and named it NQO2 [39] In 1997, Zhao et al demonstrated that NQO2 was indeed the flavoprotein discovered more than 30 years before [56] Jaiswal’s group then developed NQO2-null mice models to investigate the role of the second human QR in regulation of p53 and found that loss of NQO2 significantly decreases the level of p53 [39] Co-immunoprecipitation studies revealed a physical interaction of NQO2 with p53, indicating that an increased amount of cytosolic NQO2 protects p53 from 20S proteasomal degradation through physical association [39] Not just transcription factors Although transcription factors appear to be prime targets for QR-regulated degradation, recent studies have also identified an enzyme – ornithine decarboxylase (ODC) Catalysing the first and rate-limiting step in the polyamine biosynthesis pathway, ODC is one of the most labile cellular proteins [57] The polyamines spermidine, spermine and their precursor putrescine are abundant polycations that are present in all living cells Polyamines are essential for cellular proliferation and are involved in regulating additional fundamental cellular processes such as cellular transformation and differentiation [35] In its active form, ODC is a homodimer with two enzymatic active sites catalyzing the decarboxylation of ornithine to putrescine [58] The cellular level of ODC and its activity need to be strictly controlled because polyamines act as a double-edged sword On the one hand, they are absolutely required for maintaining growth, whereas, on the other hand, excessive levels are cytotoxic ODC degradation is FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4317 Quinone reductase as regulator of the proteasome S Sollner and P Macheroux mediated by interaction with a polyamine-induced protein termed antizyme [57] Association of antizyme with ODC subunits triggers disruption of ODC homodimers and the formation of enzymatically inactive ODC–antizyme heterodimers [59] Both in vivo and in vitro studies have indicated that ODC degradation by the 26S proteasome requires interaction with antizyme, but not ubiquitination However, recent studies revealed that there is a second, ubiquitin-independent degradation pathway for ODC that is regulated by NQO1 The QR was shown to protect ODC against proteasomal degradation both in vivo and in vitro [60] Disruption of NQO1 binding under several conditions such as oxidative stress or upon exposure to dicoumarol, a competitive inhibitor of NQO1, results in ubiquitin-independent 20S proteasomal degradation of ODC Notably, only ODC monomers are degraded by this pathway Thus, the role of antizyme in this process, if any, is confined to the ODC monomerization step ODC monomerization is also obligatory for the antizyme-independent degradation of ODC in vitro An ODC mutant that fails to dimerize is susceptible to 20S proteasomal degradation, but not to degradation by the 26S proteasome Interaction with NQO1 protects monomeric ODC from this degradation pathway, whereas inhibition of NQO1 dissociates the complex and promotes ODC degradation [35,61] Although specific mechanisms mediate the recognition of proteins destined for degradation by the 26S proteasome, it is not yet clear how proteins are recognized for degradation by the 20S core particle Recent studies have suggested that unstructured proteins such as a-synuclein and p21cip are intrinsically unstable because of their capacity to enter the 20S proteasome pore [62,63] Even a segment of unstructured region within a protein might be sufficient to direct a protein to 20S proteasomal degradation Analysis of the ODC sequence using different computational prediction algorithms indicates that ODC contains several unstructured regions Similarly, > 80% of transcription factors have been reported to possess extended regions of intrinsic disorder [64] From mammalian cells to yeast: a homologous system in a unicellular organism All of the initial studies indicating a role for QR in stabilizing transcription factors and tumour suppressors were performed with cells from a narrow range of multicellular eukaryotic organisms, i.e mammalian cells of human or murine origin Until recently, it was unclear whether the QR-operated regulation of protein 4318 degradation discovered in mammalian cells was conserved in all eukaryotes or is a recent addition to the arsenal of regulatory mechanisms found in higher multicellular eukaryotes such as mammals This issue could be partially resolved because studies with baker’s yeast (S cerevisiae) have unambiguously demonstrated that Lot6p, a homologue of mammalian NQO1, binds to the 20S proteasome and forms a ternary complex with Yap4p, a member of the yeast activator protein family of transcription factors [34] Yap4p (CIN5p, Hal6p, YOR028Cp) has been shown to increase sodium and lithium tolerance upon overexpression [65] and to confer resistance to cisplatin, a chemotherapy drug [66] In the yeast system, binding of Yap4p to the proteasome–QR complex depends on the redox state of the flavin cofactor present in the active site of Lot6p Recruitment of the transcription factor occurs when the flavin is in its fully reduced form This process is independent of the mode of reduction – either by addition of NADH or by light – and suggests that the change in redox state governs the association of Yap4p and Lot6p Although it appears likely that the redox state of the FAD cofactor of NQO1 and NQO2 plays a similar role in the mammalian system, unequivocal experimental evidence is not available at present However, the observation that recruitment of transcription factors occurs only in the presence of NADH is consistent with a redox-controlled process Further biochemical studies with Lot6p have shown that the native dimeric quaternary structure is a prerequisite for formation of the ternary complex: in its monomeric form, Lot6p still binds to the proteasome, but is no longer able to recruit the transcription factor to the complex Degradation of Yap4p by the 20S proteasome is prevented by the formation of a ternary protein complex consisting of the 20S core particle, reduced Lot6p and Yap4p Interestingly, formation of this ternary protein complex not only prevents degradation of Yap4p, but also influences the localization of the transcription factor In normal, unstressed yeast cells, Yap4p is present in the cytosol, whereas under oxidative stress it relocates to the nucleus Apparently, oxidation of the flavin cofactor of Lot6p results in the dissociation and concomitant relocalization of the released transcription factor to the nucleus where expression of stress related genes then occurs Taken together, several studies investigating transcription factors from mammalian to yeast cells, as well as regulatory proteins such as ODC, suggest that short-lived proteins are intrinsically prone to degradation by the 20S proteasome The association of a QR (NQO1, NQO2 or Lot6p) with the 20S proteasome, together with its ability to regulate the stability, and in FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS S Sollner and P Macheroux the case of the yeast system, even the localization, of those short-lived proteins suggests that QRs might play a general and central role in regulating the metabolic stability of a subset of cellular proteins Quinone reductase as regulator of the proteasome A Molecular mechanism of interaction Protection against 20S proteasomal degradation relies on the recognition of transcription factors and perhaps other intrinsically unstable proteins such as ODC by the QR The active site of the enzyme with its flavin cofactor – FAD in the case of the mammalian enzymes and FMN in the case of the homologous Lot6p – is clearly essential for the interaction, as documented by the effect of dicoumarol, a potent competitive inhibitor that pi-stacks on top of the isoalloxazine ring system, preventing association with p53 [44,67] Similarly, removal of the flavin in Lot6p disables the interaction with the yeast transcription factor Yap4p and the 20S proteasome [34] Moreover, both the presence of the cofactor and its redox state appear to be relevant In the yeast system, only reduced QR is able to recruit transcription factor Yap4p Although clear evidence is currently available only for the yeast system, studies with the mammalian system have also indicated the necessity to reduce the flavin cofactor in order to enable interaction with target proteins such as transcription factors p53 and p73a [44,45] Thus, it can be concluded that transcription factors (and probably intrinsically unstructured proteins such as ODC) bind to or near the flavin site of the QR What structural changes occur upon reduction of the flavin cofactor and how are these sensed by potential interaction partners? In principle, reduction of the isoalloxazine ring system converts N(5) from a hydrogen-bond acceptor to a hydrogen-bond donor (Fig 2) In other words, reduction of the isoalloxazine ring system may cause reorganization of hydrogen-bond interactions with neighbouring amino acids, which in turn may lead to local structural changes in the protein An instructive example for such a restructuring is given by the X-ray analysis of oxidized and reduced flavodoxin from Clostridium beijerinckii [68] In the oxidized state, C@O of Gly57 points away from N(5) of the isoalloxazine ring system Upon reduction, the C@O turns towards the N(5) position to form a hydrogen bond As a result, Gly57 adopts a different conformation and this ‘peptide flip’ also causes the movement of some amino acid side chains (Fig 2) [68] As mentioned in the introduction, QRs also adopt a flavodoxin-fold and the isoalloxazine ring engages in a similar interaction with a peptide chain In the reported structure of oxidized Lot6p, the backbone amide group of Asn96 forms a B Fig Structural changes occurring in flavodoxin upon reduction of the flavin cofactor (A) Overall structure of oxidized flavodoxin from Clostridium beijerinckii (PDB code 5nll) The FMN cofactor is shown as a colour-coded stick model (carbon, yellow; nitrogen, blue; oxygen, red) The box indicates the area where structural changes occur upon reduction of the flavin (B) Close-up of the flavin cofactor and the loop close to the flavin N(5) for both the oxidized (PDB code 5nll) and reduced (PDB code 5ull) form of flavodoxin Amino acid residues are depicted as colour-coded stick model with carbons from the oxidized form coloured grey and carbons from the reduced form coloured green In the oxidized state, C=O of Gly57 points away from N(5) of the isoalloxazine ring system Upon reduction, the C=O turns towards the N(5) position to form a hydrogen bond Consequently, Gly57 adopts a different conformation and this ‘peptide flip’ also causes movement of some amino acid side chains hydrogen bond to N(5) Upon reduction, N(5) will be protonated and hence this interaction is no longer feasible, and it is conceivable that this leads to a conformational change similar to that observed in flavodoxins Interestingly, structural comparison of QRs (human NQO1, human NQO2, mouse NQO1 and yeast Lot6p; Fig 3) shows the conservation of large hydrophobic amino acid residues (i.e conservation of a Trp residue; Fig 3E) in the peptide segment that runs along the N(5)–C(4)=O edge of the isoalloxazine ring system It is conceivable that a similar peptide flip occurs in QRs upon reduction, which then results in a repositioning of these large hydrophobic side chains such that interaction with transcription factors is enabled Interestingly, utilization of hydrophobic resi- FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4319 Quinone reductase as regulator of the proteasome A B C S Sollner and P Macheroux D Fig Comparison of the loops close to the flavin N(5) of several quinone reductases (A) NQO1 from Homo sapiens (PDB code 1d4a) (B) NQO2 from H sapiens (PDB code 1qr2) (C) NQO1 from Mus musculus (PDB code 1dxq) (D) Lot6p from Saccharomyces cerevisiae (PDB code 1t0i) Both the flavin cofactor (carbon, yellow; nitrogen, blue; oxygen, red) and the amino acid residues (carbon, grey; nitrogen, blue; oxygen, red) are depicted as colour-coded stick model (E) Sequence alignment of the loop regions shown in Fig 3A–D h NQO1, human NQO1; h NQO2, human NQO2; m NQO1, mouse NQO1; y Lot6p, yeast Lot6p E dues for the recognition and binding of unfolded protein substrates is a very common mechanism employed by chaperones [69] For example, a-crystallin, a prominent member of the small heat shock protein family, was proposed to suppress the aggregation of other proteins through an interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded protein substrates [70] Thus, it is conceivable that a similar mechanism is used by QRs for sensing and binding of intrinsically unfolded proteins such as transcription factors This mechanism could be probed by combined structure– function analysis of oxidized and reduced QR and mutagenesis of conserved residues As far as the transcription factor p53 is concerned, several amino acid residues have been implicated in the interaction with NQO1 This tumour suppressor is mutated in > 50% of human cancers [43], with Arg175His, Arg248His and Arg273His being the most frequent ‘hot-spot’ mutants [71] Mutations in the p53 gene often result in the accumulation of p53 protein variants in human cancer cells [72] Asher and coworkers investigated whether those common mutations may have an effect on binding of p53 to NQO1 They showed that the most frequent p53 variants in human cancer mentioned above were resistant to dicoumarolinduced degradation (unlike wild-type p53), probably 4320 Fig Transcription factor p53 in complex with DNA The p53 monomer is depicted as a cartoon (light blue), DNA is shown as purple stick model (PDB code 2ahi) Residues probably involved in binding to NQO1 (Arg248, Arg273) are shown as colour-coded sticks (carbon, cyan; nitrogen, blue; oxygen, red) FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS S Sollner and P Macheroux because of increased binding to NQO1, but remained sensitive to Mdm2–ubiquitin-mediated degradation Hence they concluded that NQO1 plays a major role in stabilizing p53 hot-spot mutants in human cancer cells [73] However, it needs to be taken into consideration that some variants of p53 not only lose their function, but also adopt a fold different to wild-type protein [74] In addition, p53 possesses extensive unstructured regions in its native state [75] Thus, it cannot be ruled out that the observed effect of p53 hot-spot variants on association with NQO1 is attributable to an altered conformation of the p53 variant that is different from the wild-type one However, their finding implies that the two alternative pathways for p53 degradation, the NQO1 dependent and the Mdm2 dependent, must have different p53 structural requirements Crystal structures of complexes between the core domain of human p53 and DNA half-sites reveal that two of the three residues mentioned above (Arg248, Arg273) are located at the interface of p53 and the DNA helix [76,77], indicating that the same residues that are involved in DNA recognition and binding are actually responsible for association of p53 with NQO1 (Fig 4) Conclusions and open questions The last decade has witnessed accumulating evidence for a role of eukaryotic QRs in regulating the 20S proteasomal degradation of certain transcription factors (e.g p53, Yap4p) and possibly proteins possessing a high degree of unstructured segments (e.g ODC) The body of information available clearly indicates that this pathway is relevant for the cell and complements other pathways such as ubiquitin-dependent 26S proteasomal degradation mediated by Mdm2 The concept of protecting a protein by ‘hiding it near the lion’s den’ (the catalytic chamber of the proteasome) is at first unexpected However, the proteasome represents ˚ an enormous surface (213 210 A2) [78] that offers itself for extensive protein–protein interactions and perhaps the interaction of QR and the 20S proteasome is just one example of many others still to be discovered Several issues remain unclear As discussed above, structural and biochemical information on how the recognition and binding between the various players occurs is lacking Although NAD(P)H is the most likely reducing agent for QR, it is not clear how the flavin is reoxidized, or in other words by which chemical messenger (a quinone?) transcription factors are released from their protecting protein complex Following this event, the transcription factor must rapidly relocate to the nucleus or else be degraded by the Quinone reductase as regulator of the proteasome 20S or 26S proteasome Again, the mechanism of relocalization remains obscure Finally, we not know whether our list of proteins that are subject to protection by binding to QRs is complete Are there other transcription factors and proteins in eukaryotes? And what does that tell us about their cellular function? 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the initial studies indicating a role for QR in stabilizing transcription... metabolic stability of a subset of cellular proteins Quinone reductase as regulator of the proteasome A Molecular mechanism of interaction Protection against 20S proteasomal degradation relies... naturally occurring quinones comprising vitamin K derivatives (menaquinone and phylloquinone), coenzyme Q (ubiquinone) and dopaquinone have also been shown to be substrates for mammalian QRs [13]