Differential squamous cell fates elicited by NRF2 gain of function versus KEAP1 loss of function.

Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 (NFE2L2) is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that caused by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we aspire to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer driver mutant TRP53R172H. Concomitant expression of NRF2L30F and TRP53R172H results in formation of NRF2-activated ESCC-like lesions. In contrast, while squamous-cell-specific deletion of KEAP1 induces similar NRF2 hyperactivation, the loss of KEAP1 combined with expression of TRP53R172H does not elicit the formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappear from the esophageal epithelium over time. These findings demonstrate that, while cellular NRF2 levels are similarly induced, NRF2 gain of function and KEAP1 loss of function elicits distinct fates of squamous cells. The NRF2L30F mutant mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels.

Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Supersulfides support bone growth by promoting chondrocyte proliferation in the growth plates.

OBJECTIVES: While chondrocytes have mitochondria, they receive little O2 from the bloodstream. Sulfur respiration, an essential energy production system in mitochondria, uses supersulfides instead of O2. Supersulfides are inorganic and organic sulfides with catenated sulfur atoms and are primarily produced by cysteinyl tRNA synthetase-2 (CARS2). Here, we investigated the role of supersulfides in chondrocyte proliferation and bone growth driven by growth plate chondrocyte proliferation. METHODS: We examined the effects of NaHS, an HS-/H2S donor, and cystine, the cellular source of cysteine, on the proliferation of mouse primary chondrocytes and growth of embryonic mouse tibia in vitro. We also examined the effect of RNA interference acting on the Cars2 gene on chondrocyte proliferation in the presence of cystine. RESULTS: NaHS (30 µmol/L) enhanced tibia longitudinal growth in vitro with expansion of the proliferating zone of their growth plates. While NaHS (30 µmol/L) also promoted chondrocyte proliferation only under normoxic conditions (20 % O2), cystine (0.5 mmol/L) promoted it under both normoxic and hypoxic (2 % O2) conditions. Cars2 gene knockdown abrogated the ability of cystine (0.5 mmol/L) to promote chondrocyte proliferation under normoxic conditions, indicating that supersulfides produced by CARS2 were responsible for the cystine-dependent promotion of bone growth. CONCLUSIONS: The presented results indicate that supersulfides play a vital role in bone growth achieved by chondrocyte proliferation in the growth plates driven by sulfur respiration.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Persulfide Biosynthesis Conserved Evolutionarily in All Organisms.

Significance: Persulfides/polysulfides are sulfur-catenated molecular species (i.e., R-Sn-R', n > 2; R-Sn-H, n > 1, with R = cysteine, glutathione, and proteins), such as cysteine persulfide (CysSSH). These species are abundantly formed as endogenous metabolites in mammalian and human cells and tissues. However, the persulfide synthesis mechanism has yet to be thoroughly discussed. Recent Advances: We used ß-(4-hydroxyphenyl)ethyl iodoacetamide and mass spectrometry to develop sulfur metabolomics, a highly precise, quantitative analytical method for sulfur metabolites. Critical Issues: With this method, we detected appreciable amounts of different persulfide species in biological specimens from various organisms, from the domains Bacteria, Archaea, and Eukarya. By using our rigorously quantitative approach, we identified cysteinyl-tRNA synthetase (CARS) as a novel persulfide synthase, and we found that the CysSSH synthase activity of CARS is highly conserved from the domains Bacteria to Eukarya. Because persulfide synthesis is found not only with CARS but also with other sulfotransferase enzymes in many organisms, persulfides/polysulfides are expected to contribute as fundamental elements to substantially diverse biological phenomena. In fact, persulfide generation in higher organisms-that is, plants and animals-demonstrated various physiological functions that are mediated by redox signaling, such as regulation of energy metabolism, infection, inflammation, and cell death, including ferroptosis. Future Directions: Investigating CARS-dependent persulfide production may clarify various pathways of redox signaling in physiological and pathophysiological conditions and may thereby promote the development of preventive and therapeutic measures for oxidative stress as well as different inflammatory, metabolic, and neurodegenerative diseases. Antioxid. Redox Signal. 39, 983-999.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Synthesis of Sulfides and Persulfides Is Not Impeded by Disruption of Three Canonical Enzymes in Sulfur Metabolism.

Reactive sulfur species, or persulfides and polysulfides, such as cysteine hydropersulfide and glutathione persulfide, are endogenously produced in abundance in both prokaryotes and eukaryotes, including mammals. Various forms of reactive persulfides occur in both low-molecular-weight and protein-bound thiols. The chemical properties and great supply of these molecular species suggest a pivotal role for reactive persulfides/polysulfides in different cellular regulatory processes (e.g., energy metabolism and redox signaling). We demonstrated earlier that cysteinyl-tRNA synthetase (CARS) is a new cysteine persulfide synthase (CPERS) and is responsible for the in vivo production of most reactive persulfides (polysulfides). Some researchers continue to suggest that 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS), and cystathionine γ-lyase (CSE) may also produce hydrogen sulfide and persulfides that may be generated during the transfer of sulfur from 3-mercaptopyruvate to the cysteine residues of 3-MST or direct synthesis from cysteine by CBS/CSE, respectively. We thus used integrated sulfur metabolome analysis, which we recently developed, with 3-MST knockout (KO) mice and CBS/CSE/3-MST triple-KO mice, to elucidate the possible contribution of 3-MST, CBS, and CSE to the production of reactive persulfides in vivo. We therefore quantified various sulfide metabolites in organs derived from these mutant mice and their wild-type littermates via this sulfur metabolome, which clearly revealed no significant difference between mutant mice and wild-type mice in terms of reactive persulfide production. This result indicates that 3-MST, CBS, and CSE are not major sources of endogenous reactive persulfide production; rather, CARS/CPERS is the principal enzyme that is actually involved in and even primarily responsible for the biosynthesis of reactive persulfides and polysulfides in vivo in mammals.

Cysteine hydropersulfide reduces lipid peroxidation and protects against myocardial ischaemia-reperfusion injury - Are endogenous persulfides mediators of ischaemic preconditioning?

Earlier studies revealed the presence of cysteine persulfide (CysSSH) and related polysulfide species in various mammalian tissues. CysSSH has both antioxidant and oxidant properties, modulates redox-dependent signal transduction and has been shown to mitigate oxidative stress. However, its functional relevance in the setting of myocardial ischaemia-reperfusion injury (IRI) remains unknown. The present study was undertaken to (1) study the dynamics of production and consumption of persulfides under normoxic and hypoxic conditions in the heart, and (2) determine whether exogenous administration of the CysSSH donor, cysteine trisulfide (Cys-SSS-Cys) at the onset of reperfusion rescues functional impairment and myocardial damage by interfering with lipid peroxidation. Utilising a well-established ex vivo Langendorff murine model, we here demonstrate that endogenous tissue concentrations of CysSSH are upregulated when oxygen supply is compromised (global myocardial ischaemia) and rapidly restored to baseline levels upon reperfusion, suggestive of active regulation. In a separate set of experiments, exogenous administration of Cys-SSS-Cys for 10 min at the onset of reperfusion was found to decrease malondialdehyde (MDA) concentrations, formation of 4-hydroxynonenal (4-HNE) protein adducts and rescue the heart from injury. Cys-SSS-Cys also restored post-ischaemic cardiac function, improving both coronary flow and left ventricular developed pressure (LVDP). Taken together, these results support the notion that endogenous CysSSH plays an important role as a "redox preconditioning" agent to combat the oxidative insult in myocardial IRI.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Heterozygous variants in GATA2 contribute to DCML deficiency in mice by disrupting tandem protein binding.

Accumulating lines of clinical evidence support the emerging hypothesis that loss-of-function mutations of GATA2 cause inherited hematopoietic diseases, including Emberger syndrome; dendritic cell, monocyte B and NK lymphoid (DCML) deficiency; and MonoMAC syndrome. Here, we show that mice heterozygous for an arginine-to-tryptophan substitution mutation in GATA2 (G2R398W/+), which was found in a patient with DCML deficiency, substantially phenocopy human DCML deficiency. Mice heterozygous for the GATA2-null mutation (G2-/+) do not show such phenotypes. The G2R398W protein possesses a decreased DNA-binding affinity but obstructs the function of coexpressed wild-type GATA2 through specific cis-regulatory regions, which contain two GATA motifs in direct-repeat arrangements. In contrast, G2R398W is innocuous in mice containing single GATA motifs. We conclude that the dominant-negative effect of mutant GATA2 on wild-type GATA2 through specific enhancer/silencer of GATA2 target genes perturbs the GATA2 transcriptional network, leading to the development of the DCML-like phenotype. The present mouse model provides an avenue for the understanding of molecular mechanisms underlying the pathogenesis of GATA2-related hematopoietic diseases.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Methods in sulfide and persulfide research.

Sulfides and persulfides/polysulfides (R-Sn-R', n > 2; R-Sn-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H2S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H2S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis.

Heparan sulfate (HS), a highly sulfated linear polysaccharide, is involved in diverse biological functions in various tissues. Although previous studies have suggested a possible contribution of HS to the differentiation of white adipocytes, there has been no direct evidence supporting this. Here, we inhibited the synthesis of HS chains in 3T3-L1 cells using CRISPR-Cas9 technology, resulting in impaired differentiation of adipocytes with attenuated bone morphogenetic protein 4 (BMP4)-fibroblast growth factor 1 (FGF1) signaling pathways. HS reduction resulted in reduced glucose uptake and decreased insulin-dependent intracellular signaling. We then made heterozygous mutant mice for the Ext1 gene, which encodes an enzyme essential for the HS biosynthesis, specifically in the visceral white adipose tissue (Fabp4-Cre+::Ext1flox/WT mice, hereafter called Ext1Δ/WT) to confirm the importance of HS in vivo. The expression levels of transcription factors that control adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma, were reduced in Ext1Δ/WT adipocytes, which contained smaller, unilocular lipid droplets, reduced levels of enzymes involved in lipid synthesis, and altered expression of BMP4-FGF1 signaling molecules. Furthermore, we examined the impact of HS reduction in visceral white adipose tissue on systemic glucose homeostasis. We observed that Ext1Δ/WT mice showed glucose intolerance because of insulin resistance. Our results demonstrate that HS plays a crucial role in the differentiation of white adipocytes through BMP4-FGF1 signaling pathways, thereby contributing to insulin sensitivity and glucose homeostasis.

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Production of IL-17A at Innate Immune Phase Leads to Decreased Th1 Immune Response and Attenuated Host Defense against Infection with Cryptococcus deneoformans.

IL-17A is a proinflammatory cytokine produced by many types of innate immune cells and Th17 cells and is involved in the elimination of extracellularly growing microorganisms, yet the role of this cytokine in the host defense against intracellularly growing microorganisms is not well known. Cryptococcus deneoformans is an opportunistic intracellular growth fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired immune responses. In the current study, we analyzed the role of IL-17A in the host defense against C. deneoformans infection. IL-17A was quickly produced by γδT cells at an innate immune phase in infected lungs. In IL-17A gene-disrupted mice, clearance of this fungal pathogen and the host immune response mediated by Th1 cells were significantly accelerated in infected lungs compared with wild-type mice. Similarly, killing of this fungus and production of inducible NO synthase and TNF-α were significantly enhanced in IL-17A gene-disrupted mice. In addition, elimination of this fungal pathogen, Th1 response, and expression of IL-12Rß2 and IFN-γ in NK and NKT cells were significantly suppressed by treatment with rIL-17A. The production of IL-12p40 and TNF-α from bone marrow-derived dendritic cells stimulated with C. deneoformans was significantly suppressed by rIL-17A. In addition, rIL-17A attenuated Th1 cell differentiation in splenocytes from transgenic mice highly expressing TCR for mannoprotein 98, a cryptococcal Ag, upon stimulation with recombinant mannoprotein 98. These data suggest that IL-17A may be involved in the negative regulation of the local host defense against C. deneoformans infection through suppression of the Th1 response.

Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.

The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.

Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells.

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.