Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Background: The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that is considered to mediate pleiotropic biological responses such as teratogenesis, tumour promotion, epithelial hyperplasia and the induction of drug-metabolizing enzymes to environmental contaminants usually represented by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast to the role of AhR in the regulatory mechanism of xenobiotic-metabolizing enzymes, there is no direct proof that the AhR is involved in the teratogenic effects of TCDD.

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1␣.The aryl hydrocarbon (Ah) receptor nuclear translocator (Arnt) is a member of a novel transcription factor family consisting of a basic helix-loop-helix (bHLH) structural motif contiguous with a PAS domain, a designated region conserved among Per (29, 38), Arnt (28), Ah receptor (AhR) (2, 13), and Sim (7, 34). Recent molecular cloning and biochemical studies have demonstrated that upon binding with an exogenous inducer such as 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin, the AhR is translocated from the cytoplasm to the nucleus. During this process, association of the AhR with heat shock protein 90 (HSP90) (8, 35, 52) is disrupted and a heterodimer with Arnt is formed (32, 40). The AhR-Arnt complex recognizes cis-acting DNA enhancer sequences, known as xenobiotic responsive elements (XREs), which function upstream of the cytochrome P-4501A1 (CYP1A1) gene to induce transcription (17,21). In addition to the induction of drug-metabolizing enzymes including CYP1A1, the AhR-Arnt system is considered to mediate the various biological effects of dioxin-like environmental pollutants, which include teratogenesis, tumor promotion, epithelial dysplasia, and immunosuppression (37,46,51). It has recently been reported that AhR gene disruption caused impairment of the li...

To elucidate the regulatory mechanisms underlying lens development, we searched for members of the large Maf family, which are expressed in the mouse lens, and found three, c-Maf, MafB, and Nrl. Of these, the earliest factor expressed in the lens was c-Maf. The expression of c-Maf was most prominent in lens fiber cells and persisted throughout lens development. To examine the functional contribution of c-Maf to lens development, we isolated genomic clones encompassing the murine c-maf gene and carried out its targeted disruption. Insertion of the ␤-galactosidase (lacZ) gene into the c-maf locus allowed visualization of c-Maf accumulation in heterozygous mutant mice by staining for LacZ activity. Homozygous mutant embryos and newborns lacked normal lenses. Histological examination of these mice revealed defective differentiation of lens fiber cells. The expression of crystallin genes was severely impaired in the c-maf-null mutant mouse lens. These results demonstrate that c-Maf is an indispensable regulator of lens differentiation during murine development.Lens development commences in the 9.5-day-old (e9.5) mouse embryo by invagination of the lens placode to form lens pits on either side of the prospective forebrain (1, 2). Subsequently at e10.5, the lens pit forms a lens vesicle, where embryonic ectodermal cells differentiate into primary lens fiber cells. By e13.0, the primary posterior lens fiber cells grow into the lumen to eventually fill the lens vesicle. The anterior cells of the vesicle become epithelial cells and constitute the lens germinal epithelium; secondary fiber cells then differentiate from the epithelial cells after this stage. This arrangement persists throughout the lifetime of the animal, as new lens fibers are continuously regenerated (3).Differentiation of the lens involves biosynthesis of a group of fibrous lens-specific proteins called crystallins, which constitute 80 -90% of the soluble protein of the lens (4 -6). The regulation of the crystallin genes has been characterized extensively (7-10), and an enhancer for the chicken ␣A-crystallin gene has been identified (11,12). Biochemical analyses of the core region of this enhancer revealed key interacting transcription factors (13,14). Of the cis elements identified in the enhancer, the ␣CE2 sequence, which shares high similarity with the Maf responsive element (MARE 1 (15)), is crucial for its transcriptional activity. MARE-related consensus sequences have also been found in the regulatory regions of other lensspecific genes (12).Recently a new transcription factor, L-Maf, which can interact with the ␣CE2 enhancer element, was isolated from chicken lens (13). L-Maf is a member of the large Maf oncoprotein/ transcription factor family (16 -18). The Maf family factors contain a basic leucine zipper domain and bind to MARE either as homodimers or as heterodimers with other basic leucine zipper transcription factors (19). L-Maf regulates the expression of multiple lens-specific genes, and its forced expression can convert primary chick embry...

The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.