Báo cáo khoa học: New roles of flavoproteins in molecular cell biology: An unexpected role for quinone reductases as regulators of proteasomal degradation pptx

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
¨
t Graz, Institut fu
¨
r Biochemie, Austria
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 one-
electron 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 oxy-
gen, 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 con-
fusion because ‘diaphorase activity’ could be detected
in numerous biological systems. The first ‘DT-diapho-
Keywords
flavin; 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:
(Received 9 December 2008, revised
29 April 2009, accepted 4 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 proteaso-
mal degradation of transcription factors and possibly intrinsically unstruc-

tured protein has emerged. To fulfil this role, quinone reductase binds to
the core particle of the proteasome and recruits certain transcription fac-
tors 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 stabili-
zing transcription factors by acting as inhibitors of their proteasomal
degradation.
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
rase’, reported by Ernster & Navazio [3], is now
known as mammalian NAD(P)H:quinone oxidoreduc-
tase (NQO1, isozyme 1). However, the acronym NQO
has traditionally been confined to QRs from mamma-
lian 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 oxi-
doreductase [6]. The crystal structure revealed that the
fold of the N-terminal portion resembles that of flavo-
doxin, 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 sur-

rounded by helices. The FAD cofactor is noncovalent-
ly bound at the interface of the monomers, with the
redox-active isoalloxazine ring positioned at one side
of two equivalent crevices, thereby forming two identi-
cal, independent active sites [6]. Structure determi-
nation 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 oppo-
site ends of the dimer interface. Each catalytic site
forms a large cavity, lined by residues from both pep-
tide chains with the flavin isoalloxazine ring forming
the bottom [8]. Interestingly, the flavodoxin-like struc-
ture of QRs is not restricted to mammalian enzymes,
but is conserved down to yeast. Lot6p, a homologous
QR from the unicellular model organism Saccharo-
myces 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 trans-
fer 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 cellu-
lar 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.
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 do 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. Never-
theless, a number of naturally occurring quinones com-
prising vitamin K derivatives (menaquinone and
phylloquinone), coenzyme Q (ubiquinone) and dopa-
quinone 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 com-
pounds is a more recent development. An explicit

mechanism by which NQO1 might protect cells was
first provided by Iyanagi & Yamazaki [10] by distin-
guishing between flavoproteins that catalyze one-
electron reductions and those, like NQO1, that
catalyze strict two-electron reductions. Accordingly,
two-electron reduction of quinones can avert: (a) one-
electron 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 flavopro-
teins, the hydroquinone products of the NQO1 path-
way 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 partici-
pate in regulatory cellular functions in addition to
their metabolic function as an enzyme [16]. For exam-
ple, glutathione S-transferase associates with c-Jun
N-terminal kinase leading to inhibition of kinase acti-
Quinone reductase as regulator of the proteasome S. Sollner and P. Macheroux
4314 FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS
vity and modulation of signalling and cellular prolifer-

ation [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 ubitquitin-
dependent and -independent proteasomal degradation.
This role of QRs is the focus of this minireview. Before
we shed further light on this recently discovered func-
tion 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 arti-
cles [18–20].
The bulk of cellular proteins in eukaryotic cells are
degraded by the 26S proteasome. This 2.5 MDa proteo-
lytic 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 degra-
dation is initiated by selective polyubiquitination fol-
lowed 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 pro-

teins, the 20S proteasome degrades structurally abnor-
mal, misfolded or highly oxidized proteins in a
ubiquitin-independent manner under conditions of cel-
lular 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 cowork-
ers in the course of their investigation into the deg-
radation of transcription factors. They found that
the vast majority of QR 1 (NQO1) from mouse liver
extract is bound to the 20S, but not the 26S, protea-
some [30]. Similar results were obtained with extracts
from human red blood cells and with different com-
mercially 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 acti-
vator of NQO1 [32]. Furthermore, activation of the
Nrf2 pathway by treatment with 3H-1,2-dithiole-3-
thione 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 protea-
some [34]. Using the 20S proteasome and recombi-
nant 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 fla-
vin cofactor demonstrated that two QR dimers bind
to one 20S proteasome core particle (Fig. 1) [34].
Furthermore, QR lacking both flavin cofactors (apo-
Lot6p) was unable to bind to the proteasome, indi-
cating 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].
α
β
β
α
α
β
β
α
NQO1
(reduced)
Stabilization
Degradation
20S proteasome

26S proteasome
19S cap
Mdm2 dependent
polyubiquitination
Ubiquitin
19 S cap
p53
p53
p53
Fig. 1. Proposed mechanisms of ubiquitin-
dependent (right) and ubiquitin-independent
(left) degradation pathways of p53. Free p53
(red) binds to the preformed 20S protea-
some–QR (green and pale green, respec-
tively) 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.
S. Sollner and P. Macheroux Quinone reductase as regulator of the proteasome
FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4315
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-glutamyl-
protein-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 affect-
ing access to the catalytic chamber, leading to reduced
proteasomal activity [37]. In this context, it is impor-
tant 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 out-
number 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 pro-
teasomal activity is severely downregulated by binding
of QR.
Contradictory results concerning the physical associ-
ation 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 do not form a protein complex, as pro-
posed 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 mam-
malian 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
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 degra-
dation of p53 and hence lower levels of the trans-
cription factor, p53-dependent apoptosis in both
c-irradiated normal thymocytes and in myeloid leukae-
mic cells was suppressed. These effects could be pre-
vented 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 path-
way? 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 proteaso-
mal p53 degradation is Mdm2 independent. Accord-
ingly, 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-
Quinone reductase as regulator of the proteasome S. Sollner and P. Macheroux
4316 FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS
tase) genes and stabilizes p66
Shc
(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 intracellu-
lar ROS, thereby promoting apoptosis of damaged
cells [46,47]. This generation of ROS increases expres-
sion 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-indepen-
dent 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 differ-

ing 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) con-
tains an additional structural domain near its C-termi-
nus 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 inter-
action 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 p33
ING1b
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 ele-
ments that displayed oncogenic characteristics [52].
Three alternatively spliced transcripts of the ING1 gene
have been found, encoding protein variants with a pre-
dicted size of 47, 33 and 24 kDa. p33
ING1b
(
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]. Phos-
phorylation of p33
ING1b
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 p33
ING1b
is degraded by the 20S proteasome.
Further results indicate that NQO1 inhibits the degra-
dation of p33
ING1b
and that ultraviolet irradiation
stabilizes p33
ING1b
by inducing phosphorylation at
Ser126, thereby enhancing its interaction with NQO1
[55].
Protection of transcription factors through associa-
tion with a QR is not restricted to NQO1. NQO2, a
homologue of NQO1, was shown to prevent transcrip-
tion factors from being degraded as well [39]. Human
QR 2 was described for the first time in 1961 as an
unknown mammalian cytosolic FAD-dependent flavo-
protein catalyzing the oxidation of reduced N-ribosyl-
and N-alkyldihydronicotinamides by menadione and
other quinones, but not the oxidation of NADH,
NADPH or NMNH (reduced nicotinamide mono-

nucleotide). 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 regula-
tion of p53 and found that loss of NQO2 significantly
decreases the level of p53 [39]. Co-immunoprecipita-
tion 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 tar-
gets 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 homo-
dimer 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
S. Sollner and P. Macheroux Quinone reductase as regulator of the proteasome
FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4317
mediated by interaction with a polyamine-induced pro-
tein termed antizyme [57]. Association of antizyme
with ODC subunits triggers disruption of ODC homo-
dimers 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 anti-
zyme, 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 dicou-
marol, 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 pro-
cess, 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 pro-
tects monomeric ODC from this degradation pathway,
whereas inhibition of NQO1 dissociates the complex
and promotes ODC degradation [35,61].
Although specific mechanisms mediate the recogni-
tion of proteins destined for degradation by the 26S
proteasome, it is not yet clear how proteins are recog-
nized for degradation by the 20S core particle. Recent
studies have suggested that unstructured proteins such
as a-synuclein and p21
cip
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 transcrip-
tion 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
degradation discovered in mammalian cells was con-
served 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 pro-
cess 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 prere-
quisite 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 pro-
teasome 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 degra-
dation 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 tran-
scription factors from mammalian to yeast cells, as
well as regulatory proteins such as ODC, suggest that
short-lived proteins are intrinsically prone to degrada-
tion 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
Quinone reductase as regulator of the proteasome S. Sollner and P. Macheroux
4318 FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS
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.

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 tran-
scription 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 part-
ners? 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 Clos-
tridium beijerinckii [68]. In the oxidized state, C@Oof
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 ‘pep-
tide flip’ also causes the movement of some amino acid
side chains (Fig. 2) [68]. As mentioned in the introduc-
tion, QRs also adopt a flavodoxin-fold and the isoal-
loxazine 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
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 con-
formational change similar to that observed in flavo-
doxins. 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-
A
B
Fig. 2. 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; oxy-
gen, red). The box indicates the area where structural changes
occur upon reduction of the flavin. (B) Close-up of the flavin cofac-
tor 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 conforma-
tion and this ‘peptide flip’ also causes movement of some amino
acid side chains.
S. Sollner and P. Macheroux Quinone reductase as regulator of the proteasome
FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS 4319
dues for the recognition and binding of unfolded
protein substrates is a very common mechanism
employed by chaperones [69]. For example, a-crystal-
lin, a prominent member of the small heat shock pro-
tein family, was proposed to suppress the aggregation
of other proteins through an interaction between
hydrophobic patches on its surface and exposed hydro-
phobic 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 cowork-
ers 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 dicoumarol-
induced degradation (unlike wild-type p53), probably
A
B
C
D
E
Fig. 3. Comparison of the loops close to
the flavin N(5) of several quinone reducta-
ses. (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.
Fig. 4. 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).
Quinone reductase as regulator of the proteasome S. Sollner and P. Macheroux
4320 FEBS Journal 276 (2009) 4313–4324 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 consider-
ation 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 alterna-

tive pathways for p53 degradation, the NQO1 depen-
dent and the Mdm2 dependent, must have different
p53 structural requirements. Crystal structures of com-
plexes 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 pro-
teasomal 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 prote-
asomal 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 A
˚
2
) [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 chemi-
cal messenger (a quinone?) transcription factors are
released from their protecting protein complex. Fol-
lowing this event, the transcription factor must rapidly
relocate to the nucleus or else be degraded by the
20S or 26S proteasome. Again, the mechanism of
relocalization remains obscure. Finally, we do 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?
Acknowledgements
This work was supported by the Austrian Fonds zur
Fo
¨
rderung der wissenschaftlichen Forschung (FWF)
through the Doktoratskolleg ‘Molecular Enzymology’
W901-B05 to PM.
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