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\begin{document}

\title{NRF2 signaling in cytoprotection and metabolism}



\author[1]{Shohei Murakami}%
\author[1]{Yusuke Kusano}%
\author[1]{Keito Okazaki}%
\author[2]{Takaaki Akaike}%
\author[1]{Hozumi  Motohashi}%
\affil[1]{Tohoku Daigaku}%
\affil[2]{Tohoku University Graduate School of Medicine}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
The KEAP1-NRF2 system plays a central role in cytoprotection and defense
mechanisms against oxidative stress. Because KEAP1 serves as a biosensor
for electrophiles by using its reactive thiols and because NRF2 is a
transcriptional factor regulating genes involved in the sulfur-mediated
redox reactions, the KEAP1-NRF2 system has been regarded as a
sulfur-utilizing cytoprotective mechanism. NRF2 is a key regulator of
cytoprotective genes, such as antioxidant and detoxification genes, and
also to possess potent anti-inflammatory activity. NRF2 has been
recently focused as a great modifier/regulator for the cellular
metabolism and mitochondrial function. Particularly, the NRF2-mediated
regulatory mechanisms of metabolites and mitochondria has been
considered diverse, but has not been fully-clarified yet. This review
article provides an overview of the molecular mechanisms that regulate
NRF2 signaling and its cytoprotective roles, and also highlights NRF2
contribution to the cellular metabolism, particularly in the context of
mitochondrial function and newly found sulfur metabolism.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Oxygen respiration enables highly efficient energy production by
utilizing molecular oxygen, but at the same time, it causes oxidative
damage to biomolecules, which is referred to as oxidative stress. The
defense mechanism against oxidative stress is essential for the survival
of aerobic organisms, and the failure of the defense mechanism causes
serious damage to the organisms. The KEAP1 (Kelch-like ECH (erythroid
cell-derived protein with CNC homology)-associated protein 1)-NRF2
(nuclear factor erythroid 2-related factor 2) system has been discovered
as a defense mechanism against oxidative stress and plays a central role
in oxidative stress response and protection (Itoh et al., 1995; Itoh et
al., 1997; Itoh et al., 1999; Yamamoto et al., 2018). In addition, the
KEAP1-NRF2 system has been reported to contribute to various
physiological processes such as regulation of cell differentiation and
proliferation (Hochmuth et al., 2011; Mitsuishi et al., 2012; Murakami
et al., 2014; Murakami et al., 2017), inflammatory responses (Suzuki et
al., 2017; Kobayashi et al., 2017), and anti-aging (Wati et al., 2020;
Oishi et al., 2020; Zhao et al., 2022).

Being a transcriptional activator responsible for the cellular redox
regulation, NRF2 directly enhances the expression of many antioxidant
proteins and enzymes that regulate biochemical reactions. NRF2 is
regarded as a master regulator of redox metabolism, coordinately
inducing genes encoding enzymes of glutathione system and thioredoxin
system as well as NADPH production. In addition, contribution of NRF2 to
the mitochondrial function has been also suggested (Dinkova-Kostova \&
Abramov, 2015; Holmstr\selectlanguage{ngerman}öm et al., 2016; Kasai et al., 2019).
Mitochondrial are essential for aerobic organisms to be better adapt to
the oxidative atmosphere in the current earth environment and also have
been an attractive target for the development of therapeutic agents for
various diseases, especially aging-related diseases. Recetnly, it has
been suggested that sulfur metabolism, especially cysteine metabolism,
plays an important role in mitochondrial energy production (Akaike et
al., 2017). Interestingly, the KEAP1-NRF2 system has also been shown to
regulate cellular cysteine uptake and metabolism, suggesting that it is
involved in mitochondrial energy metabolism via regulation of sulfur
metabolism (Alam et al., 2023). In this review article, we overview
recent advances in the molecular mechanisms regulating NRF2 signaling
and its contribution to metabolism and introduce important roles of
sulfur metabolism in the regulation of mitochondrial functions dependent
on the KEAP1-NRF2 system.

\textbf{Physiological roles of the KEAP1-NRF2 system}

Piles of studies have been published regarding cytoprotective function
of NRF2 in various organisms including \emph{C. elegans}
,\emph{Drosophila} , mouse, and human (Figure 1). NRF2-deficient mice
are susceptible to various exogenous stresses, while they are healthy
and fertile in a well-controlled and protected environment in a breeding
facility for experimental animals. The vulnerability and susceptibility
of the NRF2-deficient mice underscore the critical contribution of NRF2
to the response and adaptation to the environmental factors. For
example, Nrf2-mediated protection from electrophilic toxicants, such as
cigarette smoke (Rangasamy et al., 2004; Iizuka et al, 2005), and
ultraviolet from the sun (Hirota et al., 2005) has been reported in
mouse models. CncC, which is a \emph{Drosophila} orthologue of NRF2,
also confers resistance to the lethal effects of the pesticide malathion
(Misra et al., 2011). SKN-1, which is a \emph{C. elegans} orthologue of
NRF2, plays beneficial roles in the survival in the presence of
oxidative stress generated from paraquat (An \& Blackwell, 2003).

In addition to the exogenous environmental factors, NRF2 is also
important for the protection from endogenously-generated redox
perturbations. In mouse models, NRF2 protects renal cells from reactive
oxygen species (ROS) during ischemia-reperfusion injury (Liu et al.,
2009; Son et al., 2010; Ashrafian et al., 2012; Nezu et al., 2017),
pancreatic beta-cells from proteotoxicity under pathological conditions
(Lee et al., 2012; Yagishita et al., 2014; Amin et al., 2021) and
neuronal cells from neurodegenerative disorders (Pajares et al., 2016;
Rojo et al., 2018; Uruno et al., 2020). Furthermore, inflammation is one
of the major causes for the redox disturbance of endogenous origin. NRF2
exerts potent anti-inflammatory function by accelerating resolution of
acute inflammation (Itoh et al., 2004; Mochizuki et al., 2005) as well
as alleviating chronic inflammation (Suzuki et al., 2017).

Consistent with the antioxidant and anti-inflammatory functions,
anti-aging effects of NRF2 activation have been observed in mice (Wati
et al., 2020; Oishi et al., 2020; Zhao et al., 2022)
and\emph{Drosophila} (Sykiotis \& Bohmann, 2008). In mouse salivary
glands during physiological aging, age-related alterations including
fibrosis, immune cells infiltration, cell senescence, DNA damage and
lipid peroxide accumulation are all suppressed by NRF2 activation (Wati
et al., 2020). Age-related hearing loss is also delayed in\emph{Keap1}
-knockdown mice, in which NRF2 is systemically activated (Oishi et al.,
2020). In addition to the effects on age-related functional decline,
NRF2 activation by KEAP1 inhibition extends lifespan of Klotho mutant
mice, which is a progeria model (Zhao et al., 2022), and alleviates
age-related renal phenotypes, such as calcification and fibrosis. The
similar lifespan extension can be obsereved in\emph{Drosophila}
(Sykiotis \& Bohmann, 2008; Rahman et al., 2013).

Contribution of NRF2 to the health promotion in human has been also
implicated, based on the polymorphism in the promoter region
of\emph{NFE2L2} gene, which generates difference in the expression level
of NRF2. Smokers homozygous in the low expressor allele of NRF2 show
higher risk of lung cancer (Suzuki et al, 2013). People homozygous in
the high expressor allele of NRF2 show lower risk of noise-induced
hearing loss (Honkura et al., 2016). Physiological range of NRF2
activation is beneficial in principle.

\textbf{Regulatory mechanisms of NRF2-mediated transcription}

NRF2 is a potent transcriptional activator belonging to the CNC
transcription factor group (a family of bZip-type transcription factors
homologous to the \emph{Drosophila} transcription factor Cap'n'collar)
and has been originally identified as a related member of NFE2 p45 (Itoh
et al., 1995). NRF2 heterodimerizes with small MAF proteins and binds to
a consensus sequence, antioxidant response element (ARE)
(GCnnn\textsuperscript{G}/\textsubscript{C}TCA\textsuperscript{C}/\textsubscript{T})
(Motohashi et al., 2000; Motohashi et al., 2004; Katsuoka et al., 2005;
Yamamoto et al., 2006). Six functional domains, NRF2-ECH homology (Neh)
1 to 6, have been identified in NRF2 (Figure 2). The Neh1 domain
contains a basic region required for DNA binding and a bZip structure
required for heterodimer formation with small MAF proteins (Kyo et al.,
2004; Kimura et al., 2007; Kurokawa et al., 2009; Sengoku et al., 2022).
The Neh2 domain mediates NRF2 binding to KEAP1, an inhibitory regulator
of NRF2, via two motifs, DLG and ETGE motifs. The two-site binding of
NRF2 to KEAP1 homodimer enables KEAP1-CUL3 ubiquitin E3 ligase complex
to ubiquitinate NRF2 for degradation in the proteasome (Figure 3)
(Kobayashi et al., 2004; Zhang et al., 2004; Tong et al., 2006). The DLG
motif utilizes only hydrogen bonding and forms a relatively weak binding
that exhibits rapid binding and dissociation. The ETGE motif, on the
other hand, relies on both hydrogen bonding and hydrophobic interactions
to bind to KEAP1 and is thought to exhibit two-step binding and
dissociation, resulting in strong binding (Fukutomi et al., 2014; Horie
et al., 2021). The Neh3 domain was shown to interact with CHD6 for
enhancing NRF2-mediated transcriptional activation (Nioi et al., 2005).
The Neh4 and Neh5 domains are known as transcriptional activation
domains, and the binding of transcriptional coactivators, such as CREB
binding protein (CBP) / p300 and chromatin remodeling factor BRG1,
promotes transcriptional activation by NRF2 (Katoh, et al., 2001; Zhang
et al., 2007). MED16, a subunit of Mediator complex, is another binding
partner of NRF2 to the Neh4 and Neh5 domains, conferring transcriptional
activation ability by recruiting the Mediator complex and subsequently
RNA polymerase II (Sekine et al., 2015). When glucocorticoid receptor
binds to the Neh4 and Neh5 domains, NRF2 activity is suppressed (Alam et
al., 2017). The Neh6 domain contains serine residues that can be
phosphorylated by GSK3\selectlanguage{greek}β. \selectlanguage{english}NRF2 undergoes proteasome-mediated degradation
upon the phosphorylation of the Neh6 domain, indicating that the Neh6
domain mediates KEAP1-independent degradation of NRF2 (Rada et al.,
2012; Chowdhry et al., 2013).

Cooperativity with other transcription factors provides another layer of
regulation for NRF2 transcriptional activity (Figure 4). ATF4 and NRF2
cooperatively enhance expression of xCT, a cystine transporter (Ye et
al., 2014), and enzymes regulating \emph{de novo} synthesis serine
(DeNicola et al., 2015). Cooperativity of CEBPB and NRF2, which is
uniquely observed in cancer cells with persistent activation of NRF2,
promotes the enhancer activity of canonical NRF2 target genes and also
generates novel enhancers at the loci that are not normally regulated by
transiently-activated NRF2 (Okazaki et al., 2020; Okazaki et al., 2022).
The NRF2-CEBPB cooperativity is likely to underly the emergence of a
novel enhancer in \emph{NOTCH3} locus for promoting cancer stemness, and
at the same time, it also activates canonical NRF2-dependent enhancers,
such as in \emph{AKR1C1} -\emph{AKR1C2} locus, leading to the increased
chemo-resistance of cancer cells (Figure 4). Compared to the persistent
activation of NRF2, the transient activation only temporarily induces
CEBPB expression, and results in a very short duration of the
coexistence of the two factors, which hardly open the enhancers that the
NRF2-CEBPB cooperativity does. In contract, the persistent activation of
NRF2 leads to continuous expression of CEBPB, leading to constitutive
coexistence of the two factors to create a new mode of transcriptional
regulation.

KEAP1 is a substrate recognition subunit of CUL3-based ubiquitin E3
ligase and mediates NRF2 ubiquitination, serving as a negative regulator
of NRF2 (Kobayashi et al., 2004; Kobayashi et al., 2006). The most
unique feature of KEAP1 is to possess highly reactive cysteine residues,
which enables KEAP1 to serve as a biosensor for electrophiles. KEAP1
consists of three functional domains: a broad complex
tramtrack-bric-\selectlanguage{ngerman}à-brac (BTB) domain, an intervening region (IVR), a
double glycine repeat and a COOH-terminal region (DC) domain (Chauhan et
al., 2013) (Figure 2). KEAP1 forms homodimers via the BTB domain and
further forms a complex with Cullin3 (CUL3) and RING-box protein 1
(RBX1) to function as a ubiquitin E3 ligase (Figure 3). The DLG and ETGE
motifs of NRF2 interact with DC domains of the KEAP1 homodimer, which
allows ubiquitination of NRF2, resulting in proteasome-dependent
degradation of NRF2. Intriguingly, the homodimer formation of KEAP1 is
essential for the ubiquitin E3 ligase activity of KEAP1-CUL3 complex
(Suzuki et al., 2011). The enzymatic activity of KEAP1-CUL3 ubiquitin E3
ligase is inhibited when reactive cysteine residues of KEAP1, such as
Cys151, Cys273 and Cys288 in murine KEAP1, are directly modified by
electrophiles (Figure 2) (Yamamoto et al., 2008; Saito et al., 2015;
Suzuki et al., 2019). Different electrophiles target different cysteine
residues of KEAP1, which is regarded as a multimodal sensing system, and
the electrophilic signals converge on NRF2 to activate genes for
antioxidant response and cytoprotection. Thus, under normal conditions,
NRF2 is degraded and functionally repressed by KEAP1, and when
electrophiles attack KEAP1, NRF2 is de-repressed and activates
transcription. KEAP1 is an electrophilic biosensor regulating NRF2
pathway activity for the stress response.

KEAP1-independent regulation of NRF2 activity has been also described.
CUL1-\selectlanguage{greek}β\selectlanguage{english}TrCP ubiquitin E3 ligase ubiquitinates NRF2 when NRF2 is
phosphorylated at the Neh6 domain (Figure 3) (Rada et al., 2012;
Chowdhry et al., 2013). GSK3 is responsible for the NRF2 phosphorylation
at Ser344 and Ser347 in murine NRF2 (Figure 2), which is suppressed
under the active proliferation signals mediated by AKT (Taguchi et al.,
2014; Shirasaki et al., 2014).

\textbf{Metabolism regulated by NRF2}

As a key regulator of redox metabolism, NRF2 directly regulates many
enzymes and antioxidant proteins involved in the redox regulation.
Enzymes and transporters supporting glutathione synthesis and
utilization are widely regulated by NRF2, which includes catalytic and
regulatory subunits of gamma-glutamylcysteine ligase (GCLC and GCLM),
glutathione reductase (GSR), glutathione peroxidases (\emph{e.g.} ,
GPX2), glutathione-S-transferase (\emph{e.g.} , GSTM1 and GSTP1), and a
cystine transporter (xCT) (Figure 4) (Malhotra et al., 2010; Chorley et
al., 2012). Thioredoxin system is also under the regulation of NRF2. In
NRF2-activated cancer cells possessing hyperactivation of NRF2 and
consequently exhibiting NRF2 addiction, which is often caused by somatic
mutations of \emph{KEAP1} or \emph{NFE2L2} gene, glutathione synthesis
is greatly enhanced and thereby, cysteine, glutamate and glycine are
highly consumed and required for glutathione. In the NRF2-activated
cancer cells, the demand for cysteine is fulfilled by increased
expression of xCT (Sasaki et al., 2002), and the requirement of glycine
is covered by increased \emph{de novo} synthesis from serine (DeNicola
et al., 2015) and increased dependency on the uptake of extracellular
serine and glycine (LeBoeuf et al., 2020). In contrast, glutamate is
decreased and short due to glutamate export by xCT and glutamate
consumption for the glutathione synthesis, which results in the
metabolic vulnerability of NRF2-activated cancer cells (Romero et al.,
2017; Sayin et al., 2017).

Another metabolic activity regulated by NRF2 is NADPH synthesis (Figure
4). Pentose phosphate pathway contains two enzymes for the NADPH
synthesis, glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate
dehydrogenase (PGD), both of which are target genes of NRF2 (Mutsuishi
et al., 2012; Ding et al., 2021). Other NADPH synthesis steps are
regulated by isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1),
which are also regulated by NRF2 (Mutsuishi et al., 2012). Folate
metabolism-coupled NADPH production mediated by
methylenetetrahydrofolate dehydrogenase, cyclohydrolase and
formyltetrahydrofolate synthetase 1 (MTHFD1) and MTHFD2 appears to be
partly and indirectly regulated by NRF2, especially in NRF2-activated
cancer cells where NRF2 cooperates with ATF4 (Mutsuishi et al., 2012;
Fan et al., 2014).

As mentioned above, NRF2 enhances NADPH production by re-wiring
metabolism pathway, and offer strong reducing condition. The
NRF2-mediated reducing condition is beneficial for maintaining the high
efficiency of translation because many ribosomal subunits are rather
susceptible to oxidation of cysteine residues and subsequent decline of
their functionality (Chio et al., 2016). On the other hand, continuous
stabilization of NRF2 is considered to cause excessive cellular reducing
force, that is, reductive stress. Inheritable missense mutations in
small molecular weight heat-shock proteins promotes hypertrophic
cardiomyopathy by forming protein aggregate containing KEAP1, which
causes persistent activation of NRF2 (Rajasekaran et al., 2007;
Rajasekaran et al., 2011). Under this condition, NRF2-induced reductive
stress is regarded to further promotes protein aggregation, which
exacerbates cardiomyopathy, as NRF2 suppression mitigates protein
aggregation and improves the cardiac function (Kannan et al., 2013).
Comprehensive analysis of various lung cancer cell lines also showed
that NRF2 activation increases NADH vs. NAD\textsuperscript{+}, leading
to reductive stress (Weiss-Sadan et al., 2023). Consistent with these
studies, NRF2-activated cancers exhibit dependency on SLC33A1, which is
related to unfolded protein response and autophagy, possibly to avoid
protein aggregation under reductive cellular environment caused by NRF2
(Romero et al., 2020).

\textbf{Mitochondrial function and NRF2}

Mitochondria are important organelles as sites of energy metabolism in
aerobic respiration. Mitochondria contain TCA cycle as a part of glucose
catabolism, \selectlanguage{greek}β-\selectlanguage{english}oxidation pathway as a part of fatty acid catabolism,
electron transport chain (ETC), oxidative phosphorylation system and
sulfur oxidation pathway as a part of cysteine catabolism. One of the
important functions of mitochondria is to produce ATP via oxidative
phosphorylation. TCA cycle and \selectlanguage{greek}β-\selectlanguage{english}oxidation pathway provide NADH and
FADH\textsubscript{2} as substrates to the ETC that generates proton
gradient across the mitochondrial inner membrane leading to the ATP
production. Because mitochondrial dysfunction causes increased electron
leakage from the ETC and generates ROS, the KEAP1-NRF2 system has been
considered to protect cells from mitochondria-derived oxidative stress
(Dinkova-Kostova \& Abramov, 2015; Kasai et al., 2020; Esteras \&
Abranov, 2022). However, recent studies have demonstrated that the
KEAP1-NRF2 system contributes not only to mitochondrial redox regulation
but also to the regulation of energy metabolism.

NRF2 activation contributes to enhance mitochondrial function by
regulating mitochondrial biosynthesis and energy production
(Dinkova-Kostova \& Abramov, 2015; Esteras \& Abranov, 2022). For
example, NRF2 activation in skeletal muscles by KEAP1 disruption in mice
increases oxygen consumption (Uruno et al., 2016), increases myosin
heavy chain (MHC) I-positive slow fibers and thereby improves endurance
capacity during exercise (Onoki et al., 2021). Regarding mitochondria
biogenesis, NRF2 directly promotes transcription of \emph{nuclear
respiratory factor-1} (\emph{NRF-1} ), a transcription factor required
for mitochondrial biogenesis (Piantadosi et al., 2008), and NRF2
inducers have been shown to activate transcription of \selectlanguage{greek}\emph{peroxisome
proliferator-activated receptor gamma coactivator 1-α} \selectlanguage{english}(\selectlanguage{greek}\emph{PGC-1α} \selectlanguage{english})
gene, which serves as a cofactor for NRF-1 (Brose et al., 2012). As to
energy production, mitochondrial membrane potential and ATP production
are lower in \emph{Nrf2} -deficient mouse embryonic fibroblasts and
primary cultured neural cells than wild-type cells (Holmstr\selectlanguage{ngerman}öm et al.,
2013). Conversely, NRF2 activation by KEAP1 suppression increases
mitochondrial membrane potential and ATP production, suggesting that
NRF2 promotes energy production in mitochondria. In this regard, recent
transcriptome and proteome analyses revealed that factors involved in
oxidative phosphorylation and the ETC are regulated downstream of NRF2
directly or indirectly (Cho et al., 2019; Gao et al., 2020; Zhang et
al., 2021; Ryan et al., 2022). NRF2 also promotes the production of NADH
and FADH\textsubscript{2}, which are substrates of the ETC, and indeed,
the supply of these substrates is reduced in NRF2-deficient cells
(Esteras \& Abramov, 2022). Consistently, the transcriptome and proteome
analyses mentioned above demonstrated that NRF2 activation increases the
expression of enzymes related to glucose and fatty acid metabolism and
citric acid cycle enzymes involved in the production of NADH and
FADH\textsubscript{2} (Cho et al., 2019; Gao et al., 2020; Zhang et al.,
2021; Ryan et al., 2022).

\textbf{Fatty acid metabolism and NRF2-mediated mitochondrial
activation}

In mammals, fatty acids are stored in adipocytes as triglycerides,
broken down into fatty acids and glycerol as needed, and released into
the blood as free fatty acids. Fatty acids taken into the cells from the
blood undergo \selectlanguage{greek}β-\selectlanguage{english}oxidation in the mitochondria and are finally converted
to acetyl CoA, which enters the TCA cycle. In the process of
\selectlanguage{greek}β-\selectlanguage{english}oxidation, fatty acids produce more FADH\textsubscript{2} than NADH
compared with glucose, implying that energy production using fatty acids
is more dependent on Complex II of the ETC than that using glucose.

As mentioned above, disruption of \emph{Keap1} gene in mouse skeletal
muscles increases MHC I-positive slow fibers and improves exercise
endurance capacity (Onoki et al., 2021). Intriguingly, the NRF2
activation in the skeletal muscle promotes the fatty acid mobilization
and elevates succinate dehydrogenase (SDH) activity, implicating that
preferred utilization of fatty acids as energy source enhances the
NRF2-mediated endurance capacity. Consistent with the results, it was
reported that FADH\textsubscript{2} production is reduced in the hearts
of NRF2-deficient mice and that fatty acid-stimulated oxygen consumption
is increased in the mitochondria of KEAP1-knockdown mice (Ludtmann ert
al., 2014), suggesting that NRF2 activation enhances \selectlanguage{greek}β-\selectlanguage{english}oxidation.
Carnitine palmitoyl-tansferase 1 (CPT1) and CPT2 are required for the
uptake of fatty acids into the mitochondria and are rate-limiting
enzymes for fatty acid oxidation and both enzymes were also shown to be
decreased in cultured cells and livers from NRF2-deficient mice (Pang et
al., 2014; Meakin et al., 2014). The transcriptome and proteome analyses
suggest that NRF2 activation increases the expression of CPT1 and CPT2,
and in particular, CPT2 was reported to have an ARE sequence to which
NRF2 can bind, which implying that CPT2 is a direct NRF2 target gene
(Cho et al., 2019; Gao et al., 2020; Ryan et al., 2022). Furthermore, it
has been reported that NRF2 also directly promotes gene expression of
CD36, which is present in cellular and mitochondrial membranes and
involved in fatty acid transport (Maruyama et al., 2008). These
observations suggest NRF2 activation is involved in mitochondrial
membrane potential formation by promoting fatty acid uptake and fatty
acid oxidation.

\textbf{Supersulfide and NRF2}

Recently, ''supersulfides'' have been recognized as a new entity of
biomolecules (Zhang et al., 2023). Supersulfides are defined as
molecules possessing catenated sulfur, and they are present in the form
of low-molecular-weight metabolites and in the cysteine residue side
chains of proteins. Typical examples are cysteine persulfide (CysSSH)
and glutathione persulfide (GSSH) as reduced forms, and cystine
trisulfide (CysSSSCys) and glutathione trisulfide (GSSSG) as oxidized
forms. A unique chemical property of supersulfides is dual redox
reactivity to both electrophiles and nucleophiles, which enables
supersulfides to get involved in various biochemical reactions. Because
pKa value of the hydropersulfide moiety (-SSH) is lower than that of
simple thiol moiety (hydrosulfide moiety; -SH), CysSSH and GSSH are more
reactive to electrophiles such as oxidative stress than cysteine (CysSH)
and glutathione (GSH) (Ida et al., 2014). Physiological roles of
supersulfides include antioxidant functions (Ida et al., 2014; Millikin
et al., 2016), anti-inflammatory functions (Zhang et al., 2019;
Matsunaga et al., unpublished observation), and signal transduction
(Nishida et al., 2012; Nishimura et al., 2019). Supersulfides also
contribute to energy metabolism (Akaike et al., 2017; Marutani et al.,
2021; Alam et al., 2023), protein quality control (D\selectlanguage{ngerman}óka et al., 2020)
and enzymatic activity regulation (Kasamatsu et al., unpublished
observation).

Several enzymes have been identified to synthesize supersulfides.
Cystathionine \selectlanguage{greek}β-\selectlanguage{english}synthase (CBS) and cystathionine \selectlanguage{greek}γ-\selectlanguage{english}lyase (CSE) catalyze
transsulfuration, which acts as a conversion pathway of methionine into
cysteine, and also reportedly produce hydrogen sulfide/hydropersulfide
(H\textsubscript{2}S/HS\textsuperscript{--}). In addition to these
activities, CBS and CSE have been shown to possess activities to
generate CysSSH from cystine (Ida et al., 2014), which is a very unique
reaction because a disulfide bond in cystine is converted to
HS\textsuperscript{--} via C-S lyase-like reaction without consuming
reducing equivalent (Figure 5). 3-Mercaptopyruvate sulfertransferase
(3-MST) was also reported as the third enzyme generating
H\textsubscript{2}S and supersulfides (Kimura et al., 2017). However,
simultaneous disruption of the three enzymes, CBS, CSE and 3-MST, in
mice does not eliminate supersulfide production \emph{in vivo} (Zainol
Abidin et al., 2023), strongly suggesting the presence of alternative
compensatory mechanisms for supersulfide production. Indeed,
cysteinyltRNA synthetase (CARS) has been found to possess cysteine
persulfide synthesizing activity as a moonlighting function (Akaike et
al., 2017). CARS1 and CARS2 are cytoplasmic and mitochondrial isoforms,
respectively, and both isoforms possess four motifs well-conserved among
species: two of them are for zinc coordination and the other two are for
binding of pyridoxal phosphate (PLP). The former are essential for the
cysteinyl-tRNA synthesizing activity and thereby related to protein
translation, while the latter are essential for cysteine persulfide
synthesizing activity. CARS1 and CARS2 are considered to generate
low-molecular-weight supersulfides as well as to conjugate cysteine
persulfide with tRNA to generate persulfidated cysteinyl-tRNA, allowing
cysteine persulfide to be incorporated into a nascent polypeptide in the
ribosome. Although the functional significance of the protein
supersulfidation at the translation stage remains to be elucidated,
supersulfide production in mitochondria by CARS2 has been shown
essential for the mitochondrial function.

Impairment of CARS2-mediated supersulfide production depolarizes
mitochondrial membrane potential and reduces oxygen consumption (Akaike
et al., 2017; Alam et al., 2023), suggesting an essential role of
supersulfides in the mitochondrial energy metabolism. Although a
recombinant CARS protein synthesizes CysSSH, accumulation of
H\textsubscript{2}S/HS\textsuperscript{--}, rather than CysSSH, was
observed in cells. However, when mitochondria are partly depleted in the
cells by ethidium bromide treatment,
H\textsubscript{2}S/HS\textsuperscript{--} was decreased, but instead
CysSSH was increased (Akaike et al., 2017). These results unequivocally
indicate that CysSSH is reduced to
H\textsubscript{2}S/HS\textsuperscript{--} in the ETC function-dependent
manner, implying that supersulfides generated in mitochondria serve as
electron acceptors (Figure 5). The consequently-generated
H\textsubscript{2}S/HS\textsuperscript{--} is oxidized to supersulfides
by sulfide:quionone oxidoreductase (SQOR), which is considered to
prevent accumulation of H\textsubscript{2}S/HS\textsuperscript{--} and
avoid mitochondrial inhibition by sulfide toxicity (Marutani et al.,
2021). Sulfur oxidation enzymes residing in mitochondria, ETHE1 and
SUOX, also oxidize supersulfides to generate thiosulfate
(HS\textsubscript{2}O\textsubscript{3}\textsuperscript{--}), sulfite
(HSO\textsubscript{3}\textsuperscript{--}) and sulfate
(HSO\textsubscript{4}\textsuperscript{--}) using molecular oxygen
(Figure 5) (Luna-S\selectlanguage{ngerman}ánchez et al., 2017; Ziosi et al., 2017). If the
supersulfide synthesis in mitochondria is impaired, mitochondrial
electrons that should be accepted by supersulfides are expected to be
transferred to oxygen, leading to the generation of ROS. Thanks to the
presence of supersulfides, electrons leaked from the ETC are not
accepted by oxygen but by supersulfides and return to the ETC via SQOR.
The supersulfide-mediated electron flow is considered as a rescue
circuit for leaked electrons, that is, a mechanism avoiding excessive
generation of ROS and ensuring the efficiency of the ETC. Therefore, the
mitochondrial supersulfide production and the subsequent sulfur
oxidation pathway play a critical role in the mitochondrial energy
metabolism.

Consistent with the observation that sulfur metabolism makes a
substantial contribution to the mitochondrial respiration, cysteine
supply is critical for the mitochondrial activity (Alam et al., 2023).
One of the supply routes of cysteine is to uptake extracellular cystine
via a cystine transporter xCT. Another route is cysteine
intracellularly-synthesized from methionine via transsulfuration
pathway. As mentioned above, NRF2 directly activates \emph{Slc7a11}gene,
which encodes xCT (Sasaki et al., 2002), and thereby increases cellular
pool of cysteine, ultimately resulting in the increased production of
supersulfides. Importantly, NRF2-mediated mitochondrial activation is
canceled either by inhibition of xCT, suppression of CARS2-mediated
supersulfide production, or inhibition of the mitochondrial sulfur
oxidation pathway, supporting the idea that NRF2 activates mitochondria
though promoting the mitochondrial sulfur metabolism (Figure 5). From a
different point of view, the role of NRF2 in the mitochondria can be
interpreted as another mode of antioxidant function of NRF2: avoiding
excessive production of ROS and protecting cells from the oxidative
stress derived from mitochondria during oxygen respiration.

\textbf{NRF2 and supersulfides for understanding various pathogenesis}

The experimental results described above suggest that the KEAP1-NRF2
system regulates mitochondrial functions by promoting mitochondrial
biosynthesis, electron transport and fatty acid oxidation, in addition
to the previously known function of scavenging oxidative stress. NRF2
also enhances mitochondrial activity by promoting cystine uptake,
leading to the increase in the cysteine availability for the
supersulfide synthesis. This multimodal contribution of NRF2 to the
mitochondrial function underscores the requirement of the KEAP1-NRF2
system for the aerobic organisms, particularly, terrestrial life exposed
to higher concentrations of oxygen (Yumimoto et al., 2023).

In recent years, the NRF2-mediated regulation of mitochondrial function
has attracted much attention from the perspective of disease treatment.
For example, in the mouse model, NRF2 activation inhibits the
progression and exacerbation of aging-related diseases, such as
Alzheimer's disease and Parkinson's disease (Esteras et al., 2016; Uruno
et al., 2020). Mitochondrial dysfunction is one of the major causes for
these aging-related diseases, which are thought to result from a
combination of factors, such as accumulation of oxidative stress,
smoldering inflammation and impaired energy production. Therefore,
therapeutic agents that ameliorate all of these phenomena are desirable
for the treatment of the aging-related diseases, and NRF2 inducers are
expected to be ideal because various aging-related phenotypes are more
or less alleviated in the genetic model of \emph{Keap1} -knockdown mouse
in which NRF2 is activated in the whole body (Wati et al., 2020; Oishi
et al., 2020; Zhao et al., 2022). Importantly, it has been suggested
that the supersulfides are also associated with the aging-related
diseases. The supersulfides have been shown to be decreased during aging
in rodents and worms (Zivanovic et al., 2019). Particularly in the
brain, decreased protein supersulfidation is closely associated with the
progression of the pathological changes (Petrovic et al., 2021).
Supersulfides are mostly likely to serve as one of the important
downstream effectors of NRF2 for its anti-aging activity as well as the
antioxidant and anti-inflammatory functions. Further investigation will
clarify how supersulfides contribute to the human health and longevity.

\textbf{Acknowledgements}

We thank all the members in Akaike lab and Motohashi lab for their daily
efforts and achievements in establishing a new landscape of
pathophysiology regulated by the KEAP1-NRF2 system.

This work was supported by JSPS grant numbers 22K15504 (K.O.), 21H05258
(T.A., H.M.), 21H05263 (T.A.), 21H05264 (H.M.), 21H04799 (H.M.), and
22K19397 (T.A.), by JST-CREST grant number JPMJCR2024 (T.A.), and by
AMED grant number JP21zf0127001 (T.A., H.M.).



\textbf{References}

Akaike T, Ida T, Wei FY, Nishida M, Kumagai Y, Alam MM, Ihara H, Sawa T,
Matsunaga T, Kasamatsu S, Nishimura A, Morita M, Tomizawa K, Nishimura
A, Watanabe S, Inaba K, Shima H, Tanuma N, Jung M, Fujii S, Watanabe Y,
Ohmuraya M, Nagy P, Feelisch M, Fukuto JM, Motohashi H. Cysteinyl-tRNA
synthetase governs cysteine polysulfidation and mitochondrial
bioenergetics. Nat Commun. 2017 Oct 27;8(1):1177. doi:
10.1038/s41467-017-01311-y. PMID: 29079736; PMCID: PMC5660078.

Alam MM, Okazaki K, Nguyen LTT, Ota N, Kitamura H, Murakami S, Shima H,
Igarashi K, Sekine H, Motohashi H. Glucocorticoid receptor signaling
represses the antioxidant response by inhibiting histone acetylation
mediated by the transcriptional activator NRF2. J Biol Chem. 2017 May
5;292(18):7519-7530. doi: 10.1074/jbc.M116.773960. Epub 2017 Mar 17.
PMID: 28314773; PMCID: PMC5418050.

Alam MM, Kishino A, Sung E, Sekine H, Abe T, Murakami S, Akaike T,
Motohashi H. Contribution of NRF2 to sulfur metabolism and mitochondrial
activity. Redox Biol. 2023 Apr;60:102624. doi:
10.1016/j.redox.2023.102624. Epub 2023 Feb 2. PMID: 36758466; PMCID:
PMC9941419.

Amin KN, Palanisamy R, Sarada DVL, Ali D, Suzuki T, Ramkumar KM. Effect
of Rosolic acid on endothelial dysfunction under ER stress in pancreatic
microenvironment. Free Radic Res. 2021 Jun;55(6):698-713. doi:
10.1080/10715762.2021.1892090. Epub 2021 Mar 31. PMID: 33788639.

An JH, Blackwell TK. SKN-1 links C. elegans mesendodermal specification
to a conserved oxidative stress response. Genes Dev. 2003 Aug
1;17(15):1882-93. doi: 10.1101/gad.1107803. Epub 2003 Jul 17. PMID:
12869585; PMCID: PMC196237.

Ashrafian H, Czibik G, Bellahcene M, Aksentijevi\selectlanguage{polish}ć \selectlanguage{english}D, Smith AC, Mitchell
SJ, Dodd MS, Kirwan J, Byrne JJ, Ludwig C, Isackson H, Yavari A,
St\selectlanguage{ngerman}øttrup NB, Contractor H, Cahill TJ, Sahgal N, Ball DR, Birkler RI,
Hargreaves I, Tennant DA, Land J, Lygate CA, Johannsen M, Kharbanda RK,
Neubauer S, Redwood C, de Cabo R, Ahmet I, Talan M, Günther UL, Robinson
AJ, Viant MR, Pollard PJ, Tyler DJ, Watkins H. Fumarate is
cardioprotective via activation of the Nrf2 antioxidant pathway. Cell
Metab. 2012 Mar 7;15(3):361-71. doi: 10.1016/j.cmet.2012.01.017. PMID:
22405071; PMCID: PMC3314920.

Brose RD, Shin G, McGuinness MC, Schneidereith T, Purvis S, Dong GX,
Keefer J, Spencer F, Smith KD. Activation of the stress proteome as a
mechanism for small molecule therapeutics. Hum Mol Genet. 2012 Oct
1;21(19):4237-52. doi: 10.1093/hmg/dds247. Epub 2012 Jul 2. PMID:
22752410; PMCID: PMC3441123.

Chauhan N, Chaunsali L, Deshmukh P, Padmanabhan B. Analysis of
dimerization of BTB-IVR domains of Keap1 and its interaction with Cul3,
by molecular modeling. Bioinformation. 2013 May 25;9(9):450-5. doi:
10.6026/97320630009450. PMID: 23847398; PMCID: PMC3705614.

Chio IIC, Jafarnejad SM, Ponz-Sarvise M, Park Y, Rivera K, Palm W,
Wilson J, Sangar V, Hao Y, Öhlund D, Wright K, Filippini D, Lee EJ, Da
Silva B, Schoepfer C, Wilkinson JE, Buscaglia JM, DeNicola GM, Tiriac H,
Hammell M, Crawford HC, Schmidt EE, Thompson CB, Pappin DJ, Sonenberg N,
Tuveson DA. NRF2 Promotes Tumor Maintenance by Modulating mRNA
Translation in Pancreatic Cancer. Cell. 2016 Aug 11;166(4):963-976. doi:
10.1016/j.cell.2016.06.056. Epub 2016 Jul 28. PMID: 27477511; PMCID:
PMC5234705.

Cho HY, Miller-DeGraff L, Blankenship-Paris T, Wang X, Bell DA, Lih F,
Deterding L, Panduri V, Morgan DL, Yamamoto M, Reddy AJ, Talalay P,
Kleeberger SR. Sulforaphane enriched transcriptome of lung mitochondrial
energy metabolism and provided pulmonary injury protection via Nrf2 in
mice. Toxicol Appl Pharmacol. 2019 Feb 1;364:29-44. doi:
10.1016/j.taap.2018.12.004. Epub 2018 Dec 5. PMID: 30529165; PMCID:
PMC6658087.

Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, Bangura F, Xue
P, Pi J, Kleeberger SR, Bell DA. Identification of novel NRF2-regulated
genes by ChIP-Seq: influence on retinoid X receptor alpha. Nucleic Acids
Res. 2012 Aug;40(15):7416-29. doi: 10.1093/nar/gks409. Epub 2012 May 11.
PMID: 22581777; PMCID: PMC3424561.

Chowdhry S, Zhang Y, McMahon M, Sutherland C, Cuadrado A, Hayes JD. Nrf2
is controlled by two distinct \selectlanguage{greek}β-\selectlanguage{english}TrCP recognition motifs in its Neh6
domain, one of which can be modulated by GSK-3 activity. Oncogene. 2013
Aug 8;32(32):3765-81. doi: 10.1038/onc.2012.388. Epub 2012 Sep 10. PMID:
22964642; PMCID: PMC3522573.

DeNicola GM, Chen PH, Mullarky E, Sudderth JA, Hu Z, Wu D, Tang H, Xie
Y, Asara JM, Huffman KE, Wistuba II, Minna JD, DeBerardinis RJ, Cantley
LC. NRF2 regulates serine biosynthesis in non-small cell lung cancer.
Nat Genet. 2015 Dec;47(12):1475-81. doi: 10.1038/ng.3421. Epub 2015 Oct
19. Erratum in: Nat Genet. 2016 Apr;48(4):473. PMID: 26482881; PMCID:
PMC4721512.

Ding H, Chen Z, Wu K, Huang SM, Wu WL, LeBoeuf SE, Pillai RG, Rabinowitz
JD, Papagiannakopoulos T. Activation of the NRF2 antioxidant program
sensitizes tumors to G6PD inhibition. Sci Adv. 2021 Nov
19;7(47):eabk1023. doi: 10.1126/sciadv.abk1023. Epub 2021 Nov 17. PMID:
34788087; PMCID: PMC8598006.

Dinkova-Kostova AT, Abramov AY. The emerging role of Nrf2 in
mitochondrial function. Free Radic Biol Med. 2015 Nov;88(Pt B):179-188.
doi: 10.1016/j.freeradbiomed.2015.04.036. Epub 2015 May 11. PMID:
25975984; PMCID: PMC4726722.

D\selectlanguage{ngerman}óka É, Ida T, Dagnell M, Abiko Y, Luong NC, Balog N, Takata T, Espinosa
B, Nishimura A, Cheng Q, Funato Y, Miki H, Fukuto JM, Prigge JR, Schmidt
EE, Arnér ESJ, Kumagai Y, Akaike T, Nagy P. Control of protein function
through oxidation and reduction of persulfidated states. Sci Adv. 2020
Jan 1;6(1):eaax8358. doi: 10.1126/sciadv.aax8358. PMID: 31911946; PMCID:
PMC6938701.

Esteras N, Dinkova-Kostova AT, Abramov AY. Nrf2 activation in the
treatment of neurodegenerative diseases: a focus on its role in
mitochondrial bioenergetics and function. Biol Chem. 2016
May;397(5):383-400. doi: 10.1515/hsz-2015-0295. PMID: 26812787.

Esteras N, Abramov AY. Nrf2 as a regulator of mitochondrial function:
Energy metabolism and beyond. Free Radic Biol Med. 2022 Aug
20;189:136-153. doi: 10.1016/j.freeradbiomed.2022.07.013. Epub 2022 Jul
30. PMID: 35918014.

Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB, Rabinowitz JD.
Quantitative flux analysis reveals folate-dependent NADPH production.
Nature. 2014 Jun 12;510(7504):298-302. doi: 10.1038/nature13236. Epub
2014 May 4. Erratum in: Nature. 2014 Sep 25;513(7519):574. PMID:
24805240; PMCID: PMC4104482.

Fukutomi T, Takagi K, Mizushima T, Ohuchi N, Yamamoto M. Kinetic,
thermodynamic, and structural characterizations of the association
between Nrf2-DLGex degron and Keap1. Mol Cell Biol. 2014
Mar;34(5):832-46. doi: 10.1128/MCB.01191-13. Epub 2013 Dec 23. PMID:
24366543; PMCID: PMC4023822.

Gao L, Kumar V, Vellichirammal NN, Park SY, Rudebush TL, Yu L, Son WM,
Pekas EJ, Wafi AM, Hong J, Xiao P, Guda C, Wang HJ, Schultz HD, Zucker
IH. Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1-
null skeletal muscle. J Physiol. 2020 Dec;598(23):5427-5451. doi:
10.1113/JP280176. Epub 2020 Sep 23. PMID: 32893883; PMCID: PMC7749628.

Hirota A, Kawachi Y, Itoh K, Nakamura Y, Xu X, Banno T, Takahashi T,
Yamamoto M, Otsuka F. Ultraviolet A irradiation induces NF-E2-related
factor 2 activation in dermal fibroblasts: protective role in
UVA-induced apoptosis. J Invest Dermatol. 2005 Apr;124(4):825-32. doi:
10.1111/j.0022-202X.2005.23670.x. PMID: 15816842.

Hochmuth CE, Biteau B, Bohmann D, Jasper H. Redox regulation by Keap1
and Nrf2 controls intestinal stem cell proliferation in Drosophila. Cell
Stem Cell. 2011 Feb 4;8(2):188-99. doi: 10.1016/j.stem.2010.12.006.
PMID: 21295275; PMCID: PMC3035938.

Holmström KM, Baird L, Zhang Y, Hargreaves I, Chalasani A, Land JM,
Stanyer L, Yamamoto M, Dinkova-Kostova AT, Abramov AY. Nrf2 impacts
cellular bioenergetics by controlling substrate availability for
mitochondrial respiration. Biol Open. 2013 Jun 20;2(8):761-70. doi:
10.1242/bio.20134853. PMID: 23951401; PMCID: PMC3744067.

Holmström KM, Kostov RV, Dinkova-Kostova AT. The multifaceted role of
Nrf2 in mitochondrial function. Curr Opin Toxicol. 2016 Dec;1:80-91.
doi: 10.1016/j.cotox.2016.10.002. PMID: 28066829; PMCID: PMC5193490.

Honkura Y, Matsuo H, Murakami S, Sakiyama M, Mizutari K, Shiotani A,
Yamamoto M, Morita I, Shinomiya N, Kawase T, Katori Y, Motohashi H. NRF2
Is a Key Target for Prevention of Noise-Induced Hearing Loss by Reducing
Oxidative Damage of Cochlea. Sci Rep. 2016 Jan 18;6:19329. doi:
10.1038/srep19329. PMID: 26776972; PMCID: PMC4726010.

Horie Y, Suzuki T, Inoue J, Iso T, Wells G, Moore TW, Mizushima T,
Dinkova-Kostova AT, Kasai T, Kamei T, Koshiba S, Yamamoto M. Molecular
basis for the disruption of Keap1-Nrf2 interaction via Hinge \& Latch
mechanism. Commun Biol. 2021 May 14;4(1):576. doi:
10.1038/s42003-021-02100-6. PMID: 33990683; PMCID: PMC8121781.

Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, Suematsu M,
Motohashi H, Fujii S, Matsunaga T, Yamamoto M, Ono K, Devarie-Baez NO,
Xian M, Fukuto JM, Akaike T. Reactive cysteine persulfides and
S-polythiolation regulate oxidative stress and redox signaling. Proc
Natl Acad Sci U S A. 2014 May 27;111(21):7606-11. doi:
10.1073/pnas.1321232111. Epub 2014 Apr 14. PMID: 24733942; PMCID:
PMC4040604.

Iizuka T, Ishii Y, Itoh K, Kiwamoto T, Kimura T, Matsuno Y, Morishima Y,
Hegab AE, Homma S, Nomura A, Sakamoto T, Shimura M, Yoshida A, Yamamoto
M, Sekizawa K. Nrf2-deficient mice are highly susceptible to cigarette
smoke-induced emphysema. Genes Cells. 2005 Dec;10(12):1113-25. doi:
10.1111/j.1365-2443.2005.00905.x. PMID: 16324149.

Itoh K, Igarashi K, Hayashi N, Nishizawa M, Yamamoto M. Cloning and
characterization of a novel erythroid cell-derived CNC family
transcription factor heterodimerizing with the small Maf family
proteins. Mol Cell Biol. 1995 Aug;15(8):4184-93. doi:
10.1128/MCB.15.8.4184. PMID: 7623813; PMCID: PMC230657.

Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T,
Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small
Maf heterodimer mediates the induction of phase II detoxifying enzyme
genes through antioxidant response elements. Biochem Biophys Res Commun.
1997 Jul 18;236(2):313-22. doi: 10.1006/bbrc.1997.6943. PMID: 9240432.

Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto
M. Keap1 represses nuclear activation of antioxidant responsive elements
by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev.
1999 Jan 1;13(1):76-86. doi: 10.1101/gad.13.1.76. PMID: 9887101; PMCID:
PMC316370.

Itoh K, Mochizuki M, Ishii Y, Ishii T, Shibata T, Kawamoto Y, Kelly V,
Sekizawa K, Uchida K, Yamamoto M. Transcription factor Nrf2 regulates
inflammation by mediating the effect of
15-deoxy-Delta(12,14)-prostaglandin j(2). Mol Cell Biol. 2004
Jan;24(1):36-45. doi: 10.1128/MCB.24.1.36-45.2004. PMID: 14673141;
PMCID: PMC303336.

Kannan S, Muthusamy VR, Whitehead KJ, Wang L, Gomes AV, Litwin SE,
Kensler TW, Abel ED, Hoidal JR, Rajasekaran NS. Nrf2 deficiency prevents
reductive stress-induced hypertrophic cardiomyopathy. Cardiovasc Res.
2013 Oct 1;100(1):63-73. doi: 10.1093/cvr/cvt150. Epub 2013 Jun 12.
PMID: 23761402; PMCID: PMC3778956.

Kasai S, Yamazaki H, Tanji K, Engler MJ, Matsumiya T, Itoh K. Role of
the ISR-ATF4 pathway and its cross talk with Nrf2 in mitochondrial
quality control. J Clin Biochem Nutr. 2019 Jan;64(1):1-12. doi:
10.3164/jcbn.18-37. Epub 2018 Sep 15. PMID: 30705506; PMCID: PMC6348405.

Katoh Y, Itoh K, Yoshida E, Miyagishi M, Fukamizu A, Yamamoto M. Two
domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and
synergistically activate transcription. Genes Cells. 2001
Oct;6(10):857-68. doi: 10.1046/j.1365-2443.2001.00469.x. PMID: 11683914.

Katsuoka F, Motohashi H, Ishii T, Aburatani H, Engel JD, Yamamoto M.
Genetic evidence that small maf proteins are essential for the
activation of antioxidant response element-dependent genes. Mol Cell
Biol. 2005 Sep;25(18):8044-51. doi: 10.1128/MCB.25.18.8044-8051.2005.
PMID: 16135796; PMCID: PMC1234339.

Kimura M, Yamamoto T, Zhang J, Itoh K, Kyo M, Kamiya T, Aburatani H,
Katsuoka F, Kurokawa H, Tanaka T, Motohashi H, Yamamoto M. Molecular
basis distinguishing the DNA binding profile of Nrf2-Maf heterodimer
from that of Maf homodimer. J Biol Chem. 2007 Nov
16;282(46):33681-33690. doi: 10.1074/jbc.M706863200. Epub 2007 Sep 17.
Erratum in: J Biol Chem. 2015 Apr 24;290(17):10644. PMID: 17875642.

Kimura Y, Koike S, Shibuya N, Lefer D, Ogasawara Y, Kimura H.
3-Mercaptopyruvate sulfurtransferase produces potential redox regulators
cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with
signaling molecules H\textsubscript{2}S\textsubscript{2},
H\textsubscript{2}S\textsubscript{3}~and H\textsubscript{2}S. Sci Rep.
2017 Sep 5;7(1):10459. doi: 10.1038/s41598-017-11004-7. PMID: 28874874;
PMCID: PMC5585270.

Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K,
Yamamoto M. Oxidative stress sensor Keap1 functions as an adaptor for
Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol
Cell Biol. 2004 Aug;24(16):7130-9. doi:
10.1128/MCB.24.16.7130-7139.2004. PMID: 15282312; PMCID: PMC479737.

Kobayashi A, Kang MI, Watai Y, Tong KI, Shibata T, Uchida K, Yamamoto M.
Oxidative and electrophilic stresses activate Nrf2 through inhibition of
ubiquitination activity of Keap1. Mol Cell Biol. 2006 Jan;26(1):221-9.
doi: 10.1128/MCB.26.1.221-229.2006. PMID: 16354693; PMCID: PMC1317630.

Kobayashi EH, Suzuki T, Funayama R, Nagashima T, Hayashi M, Sekine H,
Tanaka N, Moriguchi T, Motohashi H, Nakayama K, Yamamoto M. Nrf2
suppresses macrophage inflammatory response by blocking proinflammatory
cytokine transcription. Nat Commun. 2016 May 23;7:11624. doi:
10.1038/ncomms11624. PMID: 27211851; PMCID: PMC4879264.

Kurokawa H, Motohashi H, Sueno S, Kimura M, Takagawa H, Kanno Y,
Yamamoto M, Tanaka T. Structural basis of alternative DNA recognition by
Maf transcription factors. Mol Cell Biol. 2009 Dec;29(23):6232-44. doi:
10.1128/MCB.00708-09. Epub 2009 Sep 21. PMID: 19797082; PMCID:
PMC2786689.

Kyo M, Yamamoto T, Motohashi H, Kamiya T, Kuroita T, Tanaka T, Engel JD,
Kawakami B, Yamamoto M. Evaluation of MafG interaction with Maf
recognition element arrays by surface plasmon resonance imaging
technique. Genes Cells. 2004 Feb;9(2):153-64. doi:
10.1111/j.1356-9597.2004.00711.x. PMID: 15009092.

LeBoeuf SE, Wu WL, Karakousi TR, Karadal B, Jackson SR, Davidson SM,
Wong KK, Koralov SB, Sayin VI, Papagiannakopoulos T. Activation of
Oxidative Stress Response in Cancer Generates a Druggable Dependency on
Exogenous Non-essential Amino Acids. Cell Metab. 2020 Feb
4;31(2):339-350.e4. doi: 10.1016/j.cmet.2019.11.012. Epub 2019 Dec 5.
PMID: 31813821; PMCID: PMC7004873.

Lee S, Hur EG, Ryoo IG, Jung KA, Kwak J, Kwak MK. Involvement of the
Nrf2-proteasome pathway in the endoplasmic reticulum stress response in
pancreatic \selectlanguage{greek}β-\selectlanguage{english}cells. Toxicol Appl Pharmacol. 2012 Nov 1;264(3):431-8.
doi: 10.1016/j.taap.2012.08.021. Epub 2012 Aug 30. PMID: 22959925.

Liu M, Grigoryev DN, Crow MT, Haas M, Yamamoto M, Reddy SP, Rabb H.
Transcription factor Nrf2 is protective during ischemic and nephrotoxic
acute kidney injury in mice. Kidney Int. 2009 Aug;76(3):277-85. doi:
10.1038/ki.2009.157. Epub 2009 May 13. PMID: 19436334.

Ludtmann MH, Angelova PR, Zhang Y, Abramov AY, Dinkova-Kostova AT. Nrf2
affects the efficiency of mitochondrial fatty acid oxidation. Biochem J.
2014 Feb 1;457(3):415-24. doi: 10.1042/BJ20130863. PMID: 24206218;
PMCID: PMC4208297.

Luna-S\selectlanguage{ngerman}ánchez M, Hidalgo-Gutiérrez A, Hildebrandt TM, Chaves-Serrano J,
Barriocanal-Casado E, Santos-Fandila Á, Romero M, Sayed RK, Duarte J,
Prokisch H, Schuelke M, Distelmaier F, Escames G, Acuña-Castroviejo D,
López LC. CoQ deficiency causes disruption of mitochondrial sulfide
oxidation, a new pathomechanism associated with this syndrome. EMBO Mol
Med. 2017 Jan;9(1):78-95. doi: 10.15252/emmm.201606345. PMID: 27856619;
PMCID: PMC5210161.

Millikin R, Bianco CL, White C, Saund SS, Henriquez S, Sosa V, Akaike T,
Kumagai Y, Soeda S, Toscano JP, Lin J, Fukuto JM. The chemical biology
of protein hydropersulfides: Studies of a possible protective function
of biological hydropersulfide generation. Free Radic Biol Med. 2016
Aug;97:136-147. doi: 10.1016/j.freeradbiomed.2016.05.013. Epub 2016 May
27. PMID: 27242269; PMCID: PMC4996688.

Malhotra D, Portales-Casamar E, Singh A, Srivastava S, Arenillas D,
Happel C, Shyr C, Wakabayashi N, Kensler TW, Wasserman WW, Biswal S.
Global mapping of binding sites for Nrf2 identifies novel targets in
cell survival response through ChIP-Seq profiling and network analysis.
Nucleic Acids Res. 2010 Sep;38(17):5718-34. doi: 10.1093/nar/gkq212.
Epub 2010 May 11. PMID: 20460467; PMCID: PMC2943601.

Marutani E, Morita M, Hirai S, Kai S, Grange RMH, Miyazaki Y, Nagashima
F, Traeger L, Magliocca A, Ida T, Matsunaga T, Flicker DR, Corman B,
Mori N, Yamazaki Y, Batten A, Li R, Tanaka T, Ikeda T, Nakagawa A,
Atochin DN, Ihara H, Olenchock BA, Shen X, Nishida M, Hanaoka K, Kevil
CG, Xian M, Bloch DB, Akaike T, Hindle AG, Motohashi H, Ichinose F.
Sulfide catabolism ameliorates hypoxic brain injury. Nat Commun. 2021
May 25;12(1):3108. doi: 10.1038/s41467-021-23363-x. PMID: 34035265;
PMCID: PMC8149856.

Maruyama A, Tsukamoto S, Nishikawa K, Yoshida A, Harada N, Motojima K,
Ishii T, Nakane A, Yamamoto M, Itoh K. Nrf2 regulates the alternative
first exons of CD36 in macrophages through specific antioxidant response
elements. Arch Biochem Biophys. 2008 Sep 1;477(1):139-45. doi:
10.1016/j.abb.2008.06.004. Epub 2008 Jun 15. PMID: 18585365.

Meakin PJ, Chowdhry S, Sharma RS, Ashford FB, Walsh SV, McCrimmon RJ,
Dinkova-Kostova AT, Dillon JF, Hayes JD, Ashford ML. Susceptibility of
Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a
high-fat diet is associated with oxidative stress, perturbation of the
unfolded protein response, and disturbance in the expression of
metabolic enzymes but not with insulin resistance. Mol Cell Biol. 2014
Sep;34(17):3305-20. doi: 10.1128/MCB.00677-14. Epub 2014 Jun 23. PMID:
24958099; PMCID: PMC4135558.

Misra JR, Horner MA, Lam G, Thummel CS. Transcriptional regulation of
xenobiotic detoxification in Drosophila. Genes Dev. 2011 Sep
1;25(17):1796-806. doi: 10.1101/gad.17280911. PMID: 21896655; PMCID:
PMC3175716.

Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H,
Yamamoto M, Motohashi H. Nrf2 redirects glucose and glutamine into
anabolic pathways in metabolic reprogramming. Cancer Cell. 2012 Jul
10;22(1):66-79. doi: 10.1016/j.ccr.2012.05.016. PMID: 22789539.

Mochizuki M, Ishii Y, Itoh K, Iizuka T, Morishima Y, Kimura T, Kiwamoto
T, Matsuno Y, Hegab AE, Nomura A, Sakamoto T, Uchida K, Yamamoto M,
Sekizawa K. Role of 15-deoxy delta(12,14) prostaglandin J2 and Nrf2
pathways in protection against acute lung injury. Am J Respir Crit Care
Med. 2005 Jun 1;171(11):1260-6. doi: 10.1164/rccm.200406-755OC. Epub
2005 Mar 4. PMID: 15750045.

Motohashi H, Katsuoka F, Shavit JA, Engel JD, Yamamoto M. Positive or
negative MARE-dependent transcriptional regulation is determined by the
abundance of small Maf proteins. Cell. 2000 Dec 8;103(6):865-75. doi:
10.1016/s0092-8674(00)00190-2. PMID: 11136972.

Motohashi H, Katsuoka F, Engel JD, Yamamoto M. Small Maf proteins serve
as transcriptional cofactors for keratinocyte differentiation in the
Keap1-Nrf2 regulatory pathway. Proc Natl Acad Sci U S A. 2004 Apr
27;101(17):6379-84. doi: 10.1073/pnas.0305902101. Epub 2004 Apr 15.
PMID: 15087497; PMCID: PMC404053.

Murakami S, Shimizu R, Romeo PH, Yamamoto M, Motohashi H. Keap1-Nrf2
system regulates cell fate determination of hematopoietic stem cells.
Genes Cells. 2014 Mar;19(3):239-53. doi: 10.1111/gtc.12126. Epub 2014
Jan 21. PMID: 24580727.

Murakami S, Suzuki T, Harigae H, Romeo PH, Yamamoto M, Motohashi H. NRF2
Activation Impairs Quiescence and Bone Marrow Reconstitution Capacity of
Hematopoietic Stem Cells. Mol Cell Biol. 2017 Sep 12;37(19):e00086-17.
doi: 10.1128/MCB.00086-17. PMID: 28674188; PMCID: PMC5599717.

Nezu M, Souma T, Yu L, Suzuki T, Saigusa D, Ito S, Suzuki N, Yamamoto M.
Transcription factor Nrf2 hyperactivation in early-phase renal
ischemia-reperfusion injury prevents tubular damage progression. Kidney
Int. 2017 Feb;91(2):387-401. doi: 10.1016/j.kint.2016.08.023. Epub 2016
Oct 24. PMID: 27789056.

Nioi P, Nguyen T, Sherratt PJ, Pickett CB. The carboxy-terminal Neh3
domain of Nrf2 is required for transcriptional activation. Mol Cell
Biol. 2005 Dec;25(24):10895-906. doi:
10.1128/MCB.25.24.10895-10906.2005. PMID: 16314513; PMCID: PMC1316965.

Nishida M, Sawa T, Kitajima N, Ono K, Inoue H, Ihara H, Motohashi H,
Yamamoto M, Suematsu M, Kurose H, van der Vliet A, Freeman BA, Shibata
T, Uchida K, Kumagai Y, Akaike T. Hydrogen sulfide anion regulates redox
signaling via electrophile sulfhydration. Nat Chem Biol. 2012
Aug;8(8):714-24. doi: 10.1038/nchembio.1018. Epub 2012 Jul 1. PMID:
22772154; PMCID: PMC4123552.

Nishimura A, Shimoda K, Tanaka T, Toyama T, Nishiyama K, Shinkai Y,
Numaga-Tomita T, Yamazaki D, Kanda Y, Akaike T, Kumagai Y, Nishida M.
Depolysulfidation of Drp1 induced by low-dose methylmercury exposure
increases cardiac vulnerability to hemodynamic overload. Sci Signal.
2019 Jun 25;12(587):eaaw1920. doi: 10.1126/scisignal.aaw1920. PMID:
31239323.

Oishi T, Matsumaru D, Ota N, Kitamura H, Zhang T, Honkura Y, Katori Y,
Motohashi H. Activation of the NRF2 pathway in Keap1-knockdown mice
attenuates progression of age-related hearing loss. NPJ Aging Mech Dis.
2020 Dec 14;6(1):14. doi: 10.1038/s41514-020-00053-4. PMID: 33318486;
PMCID: PMC7736866.

Okazaki K, Anzawa H, Liu Z, Ota N, Kitamura H, Onodera Y, Alam MM,
Matsumaru D, Suzuki T, Katsuoka F, Tadaka S, Motoike I, Watanabe M,
Hayasaka K, Sakurada A, Okada Y, Yamamoto M, Suzuki T, Kinoshita K,
Sekine H, Motohashi H. Enhancer remodeling promotes tumor-initiating
activity in NRF2-activated non-small cell lung cancers. Nat Commun. 2020
Nov 20;11(1):5911. doi: 10.1038/s41467-020-19593-0. Erratum in: Nat
Commun. 2021 Jan 15;12(1):506. PMID: 33219226; PMCID: PMC7679411.

Okazaki K, Anzawa H, Katsuoka F, Kinoshita K, Sekine H, Motohashi H.
CEBPB is required for NRF2-mediated drug resistance in NRF2-activated
non-small cell lung cancer cells. J Biochem. 2022 May 11;171(5):567-578.
doi: 10.1093/jb/mvac013. PMID: 35137113.

Onoki T, Izumi Y, Takahashi M, Murakami S, Matsumaru D, Ohta N, Wati SM,
Hatanaka N, Katsuoka F, Okutsu M, Yabe Y, Hagiwara Y, Kanzaki M, Bamba
T, Itoi E, Motohashi H. Skeletal muscle-specific Keap1 disruption
modulates fatty acid utilization and enhances exercise capacity in
female mice. Redox Biol. 2021 Jul;43:101966. doi:
10.1016/j.redox.2021.101966. Epub 2021 Apr 5. PMID: 33857757; PMCID:
PMC8050939.

Pajares M, Jiménez-Moreno N, García-Yagüe ÁJ, Escoll M, de Ceballos ML,
Van Leuven F, Rábano A, Yamamoto M, Rojo AI, Cuadrado A. Transcription
factor NFE2L2/NRF2 is a regulator of macroautophagy genes. Autophagy.
2016 Oct 2;12(10):1902-1916. doi: 10.1080/15548627.2016.1208889. Epub
2016 Jul 18. PMID: 27427974; PMCID: PMC5079676.

Pang S, Lynn DA, Lo JY, Paek J, Curran SP. SKN-1 and Nrf2 couples
proline catabolism with lipid metabolism during nutrient deprivation.
Nat Commun. 2014 Oct 6;5:5048. doi: 10.1038/ncomms6048. PMID: 25284427;
PMCID: PMC4205844.

Petrovic D, Kouroussis E, Vignane T, Filipovic MR. The Role of Protein
Persulfidation in Brain Aging and Neurodegeneration. Front Aging
Neurosci. 2021 Jun 23;13:674135. doi: 10.3389/fnagi.2021.674135. PMID:
34248604; PMCID: PMC8261153.

Piantadosi CA, Carraway MS, Babiker A, Suliman HB. Heme oxygenase-1
regulates cardiac mitochondrial biogenesis via Nrf2-mediated
transcriptional control of nuclear respiratory factor-1. Circ Res. 2008
Nov 21;103(11):1232-40. doi: 10.1161/01.RES.0000338597.71702.ad. Epub
2008 Oct 9. PMID: 18845810; PMCID: PMC2694963.

Rada P, Rojo AI, Evrard-Todeschi N, Innamorato NG, Cotte A, Jaworski T,
Tobón-Velasco JC, Devijver H, García-Mayoral MF, Van Leuven F, Hayes JD,
Bertho G, Cuadrado A. Structural and functional characterization of Nrf2
degradation by the glycogen synthase kinase 3/\selectlanguage{greek}β-\selectlanguage{english}TrCP axis. Mol Cell
Biol. 2012 Sep;32(17):3486-99. doi: 10.1128/MCB.00180-12. Epub 2012 Jul
2. PMID: 22751928; PMCID: PMC3422007.

Rahman MM, Sykiotis GP, Nishimura M, Bodmer R, Bohmann D. Declining
signal dependence of Nrf2-MafS-regulated gene expression correlates with
aging phenotypes. Aging Cell. 2013 Aug;12(4):554-62. doi:
10.1111/acel.12078. Epub 2013 May 16. PMID: 23521918; PMCID: PMC3714369.

Rajasekaran NS, Connell P, Christians ES, Yan LJ, Taylor RP, Orosz A,
Zhang XQ, Stevenson TJ, Peshock RM, Leopold JA, Barry WH, Loscalzo J,
Odelberg SJ, Benjamin IJ. Human alpha B-crystallin mutation causes
oxido-reductive stress and protein aggregation cardiomyopathy in mice.
Cell. 2007 Aug 10;130(3):427-39. doi: 10.1016/j.cell.2007.06.044. PMID:
17693254; PMCID: PMC2962423.

Rajasekaran NS, Varadharaj S, Khanderao GD, Davidson CJ, Kannan S, Firpo
MA, Zweier JL, Benjamin IJ. Sustained activation of nuclear erythroid
2-related factor 2/antioxidant response element signaling promotes
reductive stress in the human mutant protein aggregation cardiomyopathy
in mice. Antioxid Redox Signal. 2011 Mar 15;14(6):957-71. doi:
10.1089/ars.2010.3587. Epub 2011 Feb 2. PMID: 21126175; PMCID:
PMC3113450.

Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW,
Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2
enhances susceptibility to cigarette smoke-induced emphysema in mice. J
Clin Invest. 2004 Nov;114(9):1248-59. doi: 10.1172/JCI21146. PMID:
15520857; PMCID: PMC524225.

Rojo AI, Pajares M, Garc\selectlanguage{ngerman}ía-Yagüe AJ, Buendia I, Van Leuven F, Yamamoto
M, López MG, Cuadrado A. Deficiency in the transcription factor NRF2
worsens inflammatory parameters in a mouse model with combined tauopathy
and amyloidopathy. Redox Biol. 2018 Sep;18:173-180. doi:
10.1016/j.redox.2018.07.006. Epub 2018 Jul 11. PMID: 30029164; PMCID:
PMC6052199.

Romero R, Sayin VI, Davidson SM, Bauer MR, Singh SX, LeBoeuf SE,
Karakousi TR, Ellis DC, Bhutkar A, Sánchez-Rivera FJ, Subbaraj L,
Martinez B, Bronson RT, Prigge JR, Schmidt EE, Thomas CJ, Goparaju C,
Davies A, Dolgalev I, Heguy A, Allaj V, Poirier JT, Moreira AL, Rudin
CM, Pass HI, Vander Heiden MG, Jacks T, Papagiannakopoulos T. Keap1 loss
promotes Kras-driven lung cancer and results in dependence on
glutaminolysis. Nat Med. 2017 Nov;23(11):1362-1368. doi:
10.1038/nm.4407. Epub 2017 Oct 2. PMID: 28967920; PMCID: PMC5677540.

Romero R, Sánchez-Rivera FJ, Westcott PMK, Mercer KL, Bhutkar A, Muir A,
González Robles TJ, Lamboy Rodríguez S, Liao LZ, Ng SR, Li L, Colón CI,
Naranjo S, Beytagh MC, Lewis CA, Hsu PP, Bronson RT, Vander Heiden MG,
Jacks T. Keap1 mutation renders lung adenocarcinomas dependent on
Slc33a1. Nat Cancer. 2020 Jun;1(6):589-602. doi:
10.1038/s43018-020-0071-1. Epub 2020 Jun 8. Erratum in: Nat Cancer. 2020
Sep;1(9):935. PMID: 34414377; PMCID: PMC8373048.

Ryan DG, Knatko EV, Casey AM, Hukelmann JL, Dayalan Naidu S, Brenes AJ,
Ekkunagul T, Baker C, Higgins M, Tronci L, Nikitopolou E, Honda T,
Hartley RC, O'Neill LAJ, Frezza C, Lamond AI, Abramov AY, Arthur JSC,
Cantrell DA, Murphy MP, Dinkova-Kostova AT. Nrf2 activation reprograms
macrophage intermediary metabolism and suppresses the type I interferon
response. iScience. 2022 Jan 30;25(2):103827. doi:
10.1016/j.isci.2022.103827. PMID: 35198887; PMCID: PMC8844662.

Saito R, Suzuki T, Hiramoto K, Asami S, Naganuma E, Suda H, Iso T,
Yamamoto H, Morita M, Baird L, Furusawa Y, Negishi T, Ichinose M,
Yamamoto M. Characterizations of Three Major Cysteine Sensors of Keap1
in Stress Response. Mol Cell Biol. 2015 Nov 2;36(2):271-84. doi:
10.1128/MCB.00868-15. PMID: 26527616; PMCID: PMC4719294.

Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, Tamba
M, Itoh K, Yamamoto M, Bannai S. Electrophile response element-mediated
induction of the cystine/glutamate exchange transporter gene expression.
J Biol Chem. 2002 Nov 22;277(47):44765-71. doi: 10.1074/jbc.M208704200.
Epub 2002 Sep 13. PMID: 12235164.

Sayin VI, LeBoeuf SE, Singh SX, Davidson SM, Biancur D, Guzelhan BS,
Alvarez SW, Wu WL, Karakousi TR, Zavitsanou AM, Ubriaco J, Muir A,
Karagiannis D, Morris PJ, Thomas CJ, Possemato R, Vander Heiden MG,
Papagiannakopoulos T. Activation of the NRF2 antioxidant program
generates an imbalance in central carbon metabolism in cancer. Elife.
2017 Oct 2;6:e28083. doi: 10.7554/eLife.28083. PMID: 28967864; PMCID:
PMC5624783.

Sekine H, Okazaki K, Ota N, Shima H, Katoh Y, Suzuki N, Igarashi K, Ito
M, Motohashi H, Yamamoto M. The Mediator Subunit MED16 Transduces
NRF2-Activating Signals into Antioxidant Gene Expression. Mol Cell Biol.
2015 Nov 16;36(3):407-20. doi: 10.1128/MCB.00785-15. PMID: 26572828;
PMCID: PMC4719425.

Sengoku T, Shiina M, Suzuki K, Hamada K, Sato K, Uchiyama A, Kobayashi
S, Oguni A, Itaya H, Kasahara K, Moriwaki H, Watanabe C, Honma T, Okada
C, Baba S, Ohta T, Motohashi H, Yamamoto M, Ogata K. Structural basis of
transcription regulation by CNC family transcription factor, Nrf2.
Nucleic Acids Res. 2022 Nov 28;50(21):12543-12557. doi:
10.1093/nar/gkac1102. PMID: 36454022; PMCID: PMC9756947.

Shirasaki K, Taguchi K, Unno M, Motohashi H, Yamamoto M. NF-E2-related
factor 2 promotes compensatory liver hypertrophy after portal vein
branch ligation in mice. Hepatology. 2014 Jun;59(6):2371-82. doi:
10.1002/hep.27020. Epub 2014 Apr 25. PMID: 24443206.

Son TG, Camandola S, Arumugam TV, Cutler RG, Telljohann RS, Mughal MR,
Moore TA, Luo W, Yu QS, Johnson DA, Johnson JA, Greig NH, Mattson MP.
Plumbagin, a novel Nrf2/ARE activator, protects against cerebral
ischemia. J Neurochem. 2010 Mar;112(5):1316-26. doi:
10.1111/j.1471-4159.2009.06552.x. Epub 2009 Dec 17. PMID: 20028456;
PMCID: PMC2819586.

Suzuki T, Maher J, Yamamoto M. Select heterozygous Keap1 mutations have
a dominant-negative effect on wild-type Keap1 in vivo. Cancer Res. 2011
Mar 1;71(5):1700-9. doi: 10.1158/0008-5472.CAN-10-2939. Epub 2010 Dec
21. PMID: 21177379.

Suzuki T, Shibata T, Takaya K, Shiraishi K, Kohno T, Kunitoh H, Tsuta K,
Furuta K, Goto K, Hosoda F, Sakamoto H, Motohashi H, Yamamoto M.
Regulatory nexus of synthesis and degradation deciphers cellular Nrf2
expression levels. Mol Cell Biol. 2013 Jun;33(12):2402-12. doi:
10.1128/MCB.00065-13. Epub 2013 Apr 9. PMID: 23572560; PMCID:
PMC3700104.

Suzuki T, Murakami S, Biswal SS, Sakaguchi S, Harigae H, Yamamoto M,
Motohashi H. Systemic Activation of NRF2 Alleviates Lethal Autoimmune
Inflammation in Scurfy Mice. Mol Cell Biol. 2017 Jul
14;37(15):e00063-17. doi: 10.1128/MCB.00063-17. PMID: 28507037; PMCID:
PMC5514445.

Suzuki T, Muramatsu A, Saito R, Iso T, Shibata T, Kuwata K, Kawaguchi
SI, Iwawaki T, Adachi S, Suda H, Morita M, Uchida K, Baird L, Yamamoto
M. Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.
Cell Rep. 2019 Jul 16;28(3):746-758.e4. doi:
10.1016/j.celrep.2019.06.047. PMID: 31315052.

Sykiotis GP, Bohmann D. Keap1/Nrf2 signaling regulates oxidative stress
tolerance and lifespan in Drosophila. Dev Cell. 2008 Jan;14(1):76-85.
doi: 10.1016/j.devcel.2007.12.002. PMID: 18194654; PMCID: PMC2257869.

Taguchi K, Hirano I, Itoh T, Tanaka M, Miyajima A, Suzuki A, Motohashi
H, Yamamoto M. Nrf2 enhances cholangiocyte expansion in Pten-deficient
livers. Mol Cell Biol. 2014 Mar;34(5):900-13. doi: 10.1128/MCB.01384-13.
Epub 2013 Dec 30. PMID: 24379438; PMCID: PMC4023823.

Tong KI, Kobayashi A, Katsuoka F, Yamamoto M. Two-site substrate
recognition model for the Keap1-Nrf2 system: a hinge and latch
mechanism. Biol Chem. 2006 Oct-Nov;387(10-11):1311-20. doi:
10.1515/BC.2006.164. PMID: 17081101.

Uruno A, Yagishita Y, Katsuoka F, Kitajima Y, Nunomiya A, Nagatomi R, Pi
J, Biswal SS, Yamamoto M. Nrf2-Mediated Regulation of Skeletal Muscle
Glycogen Metabolism. Mol Cell Biol. 2016 May 16;36(11):1655-72. doi:
10.1128/MCB.01095-15. PMID: 27044864; PMCID: PMC4959318.

Uruno A, Matsumaru D, Ryoke R, Saito R, Kadoguchi S, Saigusa D, Saito T,
Saido TC, Kawashima R, Yamamoto M. Nrf2 Suppresses Oxidative Stress and
Inflammation in~\emph{App} ~Knock-In Alzheimer's Disease Model Mice. Mol
Cell Biol. 2020 Feb 27;40(6):e00467-19. doi: 10.1128/MCB.00467-19. PMID:
31932477; PMCID: PMC7048263.

Wati SM, Matsumaru D, Motohashi H. NRF2 pathway activation by KEAP1
inhibition attenuates the manifestation of aging phenotypes in salivary
glands. Redox Biol. 2020 Sep;36:101603. doi:
10.1016/j.redox.2020.101603. Epub 2020 Jun 12. PMID: 32590331; PMCID:
PMC7322188.

Weiss-Sadan T, Ge M, Hayashi M, Gohar M, Yao CH, de Groot A, Harry S,
Carlin A, Fischer H, Shi L, Wei TY, Adelmann CH, Wolf K, Vornbäumen T,
Dürr BR, Takahashi M, Richter M, Zhang J, Yang TY, Vijay V, Fisher DE,
Hata AN, Haigis MC, Mostoslavsky R, Bardeesy N, Papagiannakopoulos T,
Bar-Peled L. NRF2 activation induces NADH-reductive stress, providing a
metabolic vulnerability in lung cancer. Cell Metab. 2023 Apr
4;35(4):722. doi: 10.1016/j.cmet.2023.03.011. Erratum for: Cell Metab.
2023 Mar 7;35(3):487-503.e7. PMID: 37019082; PMCID: PMC10103906.

Yagishita Y, Fukutomi T, Sugawara A, Kawamura H, Takahashi T, Pi J,
Uruno A, Yamamoto M. Nrf2 protects pancreatic \selectlanguage{greek}β-\selectlanguage{english}cells from oxidative and
nitrosative stress in diabetic model mice. Diabetes. 2014
Feb;63(2):605-18. doi: 10.2337/db13-0909. Epub 2013 Nov 1. PMID:
24186865.

Yamamoto T, Kyo M, Kamiya T, Tanaka T, Engel JD, Motohashi H, Yamamoto
M. Predictive base substitution rules that determine the binding and
transcriptional specificity of Maf recognition elements. Genes Cells.
2006 Jun;11(6):575-91. doi: 10.1111/j.1365-2443.2006.00965.x. PMID:
16716189.

Yamamoto M, Kensler TW, Motohashi H. The KEAP1-NRF2 System: a
Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis.
Physiol Rev. 2018 Jul 1;98(3):1169-1203. doi:
10.1152/physrev.00023.2017. PMID: 29717933; PMCID: PMC9762786.

Ye P, Mimura J, Okada T, Sato H, Liu T, Maruyama A, Ohyama C, Itoh K.
Nrf2- and ATF4-dependent upregulation of xCT modulates the sensitivity
of T24 bladder carcinoma cells to proteasome inhibition. Mol Cell Biol.
2014 Sep 15;34(18):3421-34. doi: 10.1128/MCB.00221-14. Epub 2014 Jul 7.
Erratum in: Mol Cell Biol. 2015 Jul;35(13):2366. PMID: 25002527; PMCID:
PMC4135628.

Yumimoto K, Sugiyama S, Motomura S, Takahashi D, Nakayama KI. Molecular
evolution of Keap1 was essential for adaptation of vertebrates to
terrestrial life. Sci Adv. 2023 May 19;9(20):eadg2379. doi:
10.1126/sciadv.adg2379. Epub 2023 May 19. PMID: 37205751; PMCID:
PMC10198636.

Zainol Abidin QH, Ida T, Morita M, Matsunaga T, Nishimura A, Jung M,
Hassan N, Takata T, Ishii I, Kruger W, Wang R, Motohashi H, Tsutsui M,
Akaike T. Synthesis of Sulfides and Persulfides Is Not Impeded by
Disruption of Three Canonical Enzymes in Sulfur Metabolism. Antioxidants
(Basel). 2023 Apr 3;12(4):868. doi: 10.3390/antiox12040868. PMID:
37107243; PMCID: PMC10135671.

Zhao M, Murakami S, Matsumaru D, Kawauchi T, Nabeshima YI, Motohashi H.
NRF2 pathway activation attenuates ageing-related renal phenotypes due
to \selectlanguage{greek}α-\selectlanguage{english}klotho deficiency. J Biochem. 2022 May 11;171(5):579-589. doi:
10.1093/jb/mvac014. PMID: 35137128.

Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M. Keap1 is a
redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin
ligase complex. Mol Cell Biol. 2004 Dec;24(24):10941-53. doi:
10.1128/MCB.24.24.10941-10953.2004. PMID: 15572695; PMCID: PMC533977.

Zhang J, Hosoya T, Maruyama A, Nishikawa K, Maher JM, Ohta T, Motohashi
H, Fukamizu A, Shibahara S, Itoh K, Yamamoto M. Nrf2 Neh5 domain is
differentially utilized in the transactivation of cytoprotective genes.
Biochem J. 2007 Jun 15;404(3):459-66. doi: 10.1042/BJ20061611. PMID:
17313370; PMCID: PMC1896277.

Zhang T, Ono K, Tsutsuki H, Ihara H, Islam W, Akaike T, Sawa T. Enhanced
Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate
Lethal Endotoxin Shock. Cell Chem Biol. 2019 May 16;26(5):686-698.e4.
doi: 10.1016/j.chembiol.2019.02.003. Epub 2019 Mar 7. PMID: 30853417.

Zhang T, Akaike T, Sawa T. Redox Regulation of Xenobiotics by Reactive
Sulfur and Supersulfide Species. Antioxid Redox Signal. 2023 Jun 9. doi:
10.1089/ars.2022.0172. Epub ahead of print. PMID: 37294201.

Zhang X, Ye L, Xu H, Zhou Q, Tan B, Yi Q, Yan L, Xie M, Zhang Y, Tian J,
Zhu J. NRF2 is required for structural and metabolic maturation of human
induced pluripotent stem cell-derived ardiomyocytes. Stem Cell Res Ther.
2021 Mar 24;12(1):208. doi: 10.1186/s13287-021-02264-2. PMID: 33762018;
PMCID: PMC7992990.

Ziosi M, Di Meo I, Kleiner G, Gao XH, Barca E, Sanchez-Quintero MJ,
Tadesse S, Jiang H, Qiao C, Rodenburg RJ, Scalais E, Schuelke M, Willard
B, Hatzoglou M, Tiranti V, Quinzii CM. Coenzyme Q deficiency causes
impairment of the sulfide oxidation pathway. EMBO Mol Med. 2017
Jan;9(1):96-111. doi: 10.15252/emmm.201606356. PMID: 27856618; PMCID:
PMC5210092.

Zivanovic J, Kouroussis E, Kohl JB, Adhikari B, Bursac B, Schott-Roux S,
Petrovic D, Miljkovic JL, Thomas-Lopez D, Jung Y, Miler M, Mitchell S,
Milosevic V, Gomes JE, Benhar M, Gonzalez-Zorn B, Ivanovic-Burmazovic I,
Torregrossa R, Mitchell JR, Whiteman M, Schwarz G, Snyder SH, Paul BD,
Carroll KS, Filipovic MR. Selective Persulfide Detection Reveals
Evolutionarily Conserved Antiaging Effects of S-Sulfhydration. Cell
Metab. 2019 Dec 3;30(6):1152-1170.e13. doi: 10.1016/j.cmet.2019.10.007.
Epub 2019 Nov 14. Erratum in: Cell Metab. 2020 Jan 7;31(1):207. PMID:
31735592; PMCID: PMC7185476.

\textbf{Figure legends}

\textbf{Figure 1. Physiological roles of NRF2.}

NRF2 activates transcription of various genes involved in diverse
cytoprotective function under a physiological condition.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}



\textbf{Figure 2. Domain structures of NRF2 and KEAP1.}

NRF2 consists of 6 functional domains, Neh1 to Neh6. The function of
each Neh domain is shown on the top of the NRF2 structure and some
representative factors binding to each domain are also shown. Two serine
residues (Ser344 and Ser347) of murine NRF2 are phosphorylation target
sites of GSK3. KEAP1 has three domains. Their function is shown on the
top of the KEAP1 structure. Three cysteine residues (Cys151, Cys273 and
Cys288) of murine KEAP1 are major functional target sites of
electrophiles.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}



\textbf{Figure 3. KEAP1-dependent and KEAP1-indepdnednt degradation of
NRF2.}

KEAP1 serves as an NRF2-recognizing subunit of CUL3-based ubiquitin E3
ligase and leads NRF2 to proteasome-dependent degradation. KEAP1 is
inactivated by thiol modification with electrophiles, which allows NRF2
to be translocated to nucleus and activate its target gene
transcription. When NRF2 is phosphorylated by GSK3, \selectlanguage{greek}β\selectlanguage{english}TrCP serves as an
NRF2-recognizing subunit of CUL1-based ubiquitin E3 ligase.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}



\textbf{Figure 4. Cooperativity of NRF2 with ATF4 and CEBPB.}

Functional cooperativity of NRF2 with ATF4 and CEBPB occurs in cancer
cells with constitutive activation of NRF2. Gene categories shown in red
are canonical NRF2 target genes. Gene categories shown in green and blue
are regulated under the regulation by NRF2-ATF4 and NRF2-CEBPB
cooperativity, respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}



\textbf{Figure 5. Sulfur metabolism in mitochondria.}

Flow of sulfur metabolites in cells and mitochondria is shown with
responsible enzymes for each step. Enzymes involved in the supersulfide
production and sulfur oxidation pathway are shown in blue and green,
respectively. I, II, III and IV indicate complexes in electron transport
chain. Q and QH\textsubscript{2} indicate ubiquinone and ubiquinol,
respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\selectlanguage{english}
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