Deletion of hepatocyte cysteine dioxygenase type 1, a bile acid repressed gene, enhances glutathione synthesis and ameliorates acetaminophen hepatotoxicity.

Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans.

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Proteins are large molecules in our cells that perform various roles, from acting as channels through which nutrients can enter the cell, to forming structural assemblies that help the cell keep its shape. Proteins are formed of chains of building blocks called amino acids. There are 20 common amino acids, each with a different 'side chain' that confers it with specific features. Cysteine is one of these 20 amino acids. Its side chain has a 'thiol' group, made up of a sulfur atom and a hydrogen atom. This thiol group is very reactive, and it is an essential building block of enzymes (proteins that speed up chemical reactions within the cell), structural proteins and signaling molecules. While cysteine is an essential amino acid for the cell to function, excess cysteine can be toxic. The concentration of cysteine in animal cells is tightly regulated by an enzyme called cysteine dioxygenase. This enzyme is implicated in two rare conditions that affect metabolism, where the product of cysteine dioxygenase is a key driver of disease severity. Additionally, cysteine dioxygenase acts as a tumor suppressor gene, and its activity becomes blocked in diverse cancers. Understanding how cysteine dioxygenase is regulated may be important for research into these conditions. While it has been shown that excess cysteine drives the production and activity of cysteine dioxygenase, how the cell detects high levels of cysteine remained unknown. Warnhoff et al. sought to resolve this question using the roundworm Caenorhabditis elegans. First, the scientists demonstrated that, like in mammals, high levels of cysteine drive the production of cysteine dioxygenase in C. elegans. Next, the researchers used an approach called an unbiased genetic screening to find genes that induce cysteine dioxygenase production when they are mutated. These experiments revealed that the protein HIF-1 can drive the production of cysteine dioxygenase when it is activated by a pathway that senses hydrogen sulfide gas. Based on these results, Warnhoff et al. propose that high levels of cysteine lead to the production of hydrogen sulfide gas that in turn drives the production of cysteine dioxygenase via HIF-1 activation of gene expression. The results reported by Warnhoff et al. suggest that modulating HIF-1 signaling could control the activity of cysteine dioxygenase. This information could be used in the future to develop therapies for molybdenum cofactor deficiency, isolated sulfite oxidase deficiency and several types of cancer. However, first it will be necessary to demonstrate that the same signaling pathway is active in humans.

Enzyme-Catalyzed Oxidative Degradation of Ergothioneine.

Ergothioneine is a sulfur-containing metabolite that is produced by bacteria and fungi, and is absorbed by plants and animals as a micronutrient. Ergothioneine reacts with harmful oxidants, including singlet oxygen and hydrogen peroxide, and may therefore protect cells against oxidative stress. Herein we describe two enzymes from actinobacteria that cooperate in the specific oxidative degradation of ergothioneine. The first enzyme is an iron-dependent thiol dioxygenase that produces ergothioneine sulfinic acid. A crystal structure of ergothioneine dioxygenase from Thermocatellispora tengchongensis reveals many similarities with cysteine dioxygenases, suggesting that the two enzymes share a common mechanism. The second enzyme is a metal-dependent ergothioneine sulfinic acid desulfinase that produces Nα-trimethylhistidine and SO2 . The discovery that certain actinobacteria contain the enzymatic machinery for O2 -dependent biosynthesis and O2 -dependent degradation of ergothioneine indicates that these organisms may actively manage their ergothioneine content.

Identification of a novel enzyme and the regulation of key enzymes in mammalian taurine synthesis.

Taurine has many pharmacological roles on various tissues. The maintenance of abundant taurine content in the mammalian body through endogenous synthesis, in addition to exogenous intake, is the essential factor for morphological and functional maintenances in most tissues. The synthesis of taurine from sulfur-containing amino acids is influenced by various factors. Previous literature findings indicate the influence of the intake of proteins and sulfur-containing amino acids on the activity of the rate-limiting enzymes cysteine dioxygenase and cysteine sulfinate decarboxylase. In addition, the regulation of the activity and expression of taurine-synthesis enzymes by hormones, bile acids, and inflammatory cytokines through nuclear receptors have been reported in liver and reproductive tissues. Furthermore, flavin-containing monooxygenase subtype 1 was recently identified as the taurine-synthesis enzyme that converts hypotaurine to taurine. This review introduces the novel taurine synthesis enzyme and the nuclear receptor-associated regulation of key enzymes in taurine synthesis.

Metal Site and Size-Controlled BTC-Based MOF as Cysteine Oxidase Mimic for Self-Cascade Detection of Cysteine and Hg2.

Nanozyme-mediated strategy for sensing has been widely applied nowadays, in which the construction of a nanozyme cascade platform is an effective and challenging method to simulate the complexity and multifunctionality of natural systems. Herein, a simple and convenient self-cascade sensing platform was developed for the fluorescent detection of cysteine and Hg2+ by a BTC-based MOF through screening the metal sites and crystal sizes. By the introduction of polyvinylpyrrolidone, the as-prepared Cu-BTC possessed a metal center of Cu2+ and smaller size, which exhibited both cysteine oxidase- and peroxidase-like activities. The dual enzymic characters of Cu-BTC made a self-cascade reaction occur during which cysteine was first oxidized to cystine and generated H2O2 in the presence of O2, then H2O2 was decomposed into ·OH, and finally the ·OH triggered the turn-on fluorescence of Cu-BTC. Based on the self-cascade reactions and high affinity of Hg2+ and -SH within cysteine, a fluorescent method was developed to detect cysteine and Hg2+ with a range of 0-160/0-15 µM and a limit of detection of 0.04/0.09 µM, respectively. This work reveals the important role of the Cu2+ center for mimicking cysteine oxidase and gives a feasible strategy for constructing simple self-cascade reactions.

Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

BACKGROUND: Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. METHODS: We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. RESULTS: CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p < .05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CONCLUSIONS: CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Monitoring ADO dependent proteolysis in cells using fluorescent reporter proteins.

2-Aminoethanethiol dioxygenase (ADO) is the mammalian orthologue of the plant cysteine oxidases and together these enzymes are responsible for catalysing dioxygenation of N-terminal cysteine residues of certain proteins. This modification creates an N-degron motif that permits arginylation and subsequent proteasomal degradation of such proteins via the Arg-branch of the N-degron pathway. In humans 4 proteins have been identified as substrates of ADO; regulators of G-protein signalling (RGS) 4, 5 and 16, and interleukin-32 (IL-32). Nt-cysteine dioxygenation of these proteins occurs rapidly under normoxic conditions, but ADO activity is very sensitive to O2 availability and as such the stability of substrate proteins is inversely proportional to cellular O2 levels. Much is still to understand about the biochemistry and physiology of this pathway in vitro and in vivo, and Cys N-degron targeted fluorescent proteins can provide a simple and effective tool to study this at both subcellular and high-throughput scales. This chapter describes the design, production and implementation of a fluorescent fusion protein proteolytically regulated by ADO and the N-degron pathway.

Genome-Wide Identification and Analysis of the Plant Cysteine Oxidase (PCO) Gene Family in Brassica napus and Its Role in Abiotic Stress Response.

Plant Cysteine Oxidase (PCO) is a plant O2-sensing enzyme catalyzing the oxidation of cysteine to Cys-sulfinic acid at the N-termini of target proteins. To better understand the Brassica napus PCO gene family, PCO genes in B. napus and related species were analyzed. In this study, 20, 7 and 8 PCO genes were identified in Brassica napus, Brassica rapa and Brassica oleracea, respectively. According to phylogenetic analysis, the PCOs were divided into five groups: PCO1, PCO2, PCO3, PCO4 and PCO5. Gene organization and motif distribution analysis suggested that the PCO gene family was relatively conserved during evolution. According to the public expression data, PCO genes were expressed in different tissues at different developmental stages. Moreover, qRT-PCR data showed that most of the Bna/Bra/BoPCO5 members were expressed in leaves, roots, flowers and siliques, suggesting an important role in both vegetative and reproductive development. Expression of BnaPCO was induced by various abiotic stress, especially waterlogging stress, which was consistent with the result of cis-element analysis. In this study, the PCO gene family of Brassicaceae was analyzed for the first time, which contributes to a comprehensive understanding of the origin and evolution of PCO genes in Brassicaceae and the function of BnaPCO in abiotic stress responses.

A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Kinetic Measurements to Investigate the Oxygen-Sensing Properties of Plant Cysteine Oxidases.

Enzymatic O2 sensors transduce the availability of O2 within the cell into a physiological, typically adaptive response. One such O2-sensing enzymatic family is the N-terminal cysteine dioxygenases in plants (plant cysteine oxidases [PCOs]). In vitro kinetic studies have determined the O2-sensing capacity of PCOs. Here we describe the rationale and experimental protocol for an assay with which the O2 sensitivity of Arabidopsis thaliana PCOs (AtPCOs) can be measured. We explain each step from the recombinant protein synthesis of AtPCOs to the steady-state kinetic assays of AtPCOs for primary substrate and O2 from which kinetic parameters can be derived. The same techniques can be applied to other N-terminal cysteine thiol dioxygenases, e.g. 2-aminoethanethiol dioxygenase (ADO), and similar principles can be applied to determine kinetic characteristics of other oxygenase enzymes towards O2.

Spectroscopic analysis of the mammalian enzyme cysteine dioxygenase.

l-Cysteine (Cys) is an essential building block for the synthesis of new proteins and serves as a precursor for several biologically important sulfur-containing molecules, such as coenzyme A, taurine, glutathione, and inorganic sulfate. However, organisms must tightly regulate the concentration of free Cys, as elevated levels of this semi-essential amino acid can be extremely harmful. The non-heme iron enzyme cysteine dioxygenase (CDO) serves to maintain the proper levels of Cys by catalyzing its oxidation to cysteine sulfinic acid. Crystal structures of resting and substrate-bound mammalian CDO revealed two surprising structural motifs in the first and second coordination spheres of the Fe center. The first is the existence of a neutral three histidine (3-His) facial triad that coordinates the Fe ion, as opposed to an anionic 2-His-1-carboxylate facial triad that is typically observed in mononuclear non-heme Fe(II) dioxygenases. The second unusual structural feature exhibited by mammalian CDO is the presence of a covalent crosslink between the sulfur of a Cys residue and an ortho-carbon of a tyrosine residue. Spectroscopic studies of CDO have provided invaluable insights into the roles that these unusual features play with regards to substrate Cys and co-substrate O2 binding and activation. In this chapter, we summarize results obtained from electronic absorption, electron paramagnetic resonance, magnetic circular dichroism, resonance Raman, and Mössbauer spectroscopic studies of mammalian CDO carried out in the last two decades. Pertinent results obtained from complementary computational studies are also briefly summarized.

Testosterone enhances taurine synthesis by upregulating androgen receptor and cysteine sulfinic acid decarboxylase expressions in male mouse liver.

Taurine is an end-product of cysteine metabolism, whereas cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD) are key enzymes regulating taurine synthesis. Sex steroids, including estrogens and androgens, are associated with liver physiopathological processes; however, we still do not know whether taurine and sex steroids interact in regulating liver physiology and hepatic diseases, and whether there are sex differences, although our recent study shows that the estrogen is involved in regulating taurine synthesis in mouse liver. The present study was thus proposed to identify whether 17-ß-estradiol and testosterone (T) play their roles by regulating CDO and CSAD expression and taurine synthesis in male mouse liver. Our results demonstrated that testosterone did not have a significant influence on CDO expression but significantly enhanced CSAD, androgen receptor (AR) expressions, and taurine levels in mouse liver, cultured hepatocytes, and HepG2 cells, whereas these effects were abrogated by AR antagonist flutamide. Furthermore, our results showed that testosterone increased CSAD-promoter-luciferase activity through the direct interaction of the AR DNA binding domain with the CSAD promoter. These findings first demonstrate that testosterone acts as an important factor to regulate sulfur amino acid metabolism and taurine synthesis through AR/CSAD signaling pathway. In addition, the in vivo and in vitro experiments showed that 17-ß-estradiol has no significant effects on liver CSAD expression and taurine synthesis in male mice and suggest that the effects of sex steroids on the taurine synthesis in mouse liver have sex differences. These results are crucial for understanding the physiological functions of taurine/androgen and their interacting mechanisms in the liver.NEW & NOTEWORTHY This study demonstrates that testosterone functions to enhance taurine synthesis by interacting with androgen receptor and binding to cysteine sulfinate decarboxylase (CSAD) promoter zone. Whereas estrogen has no significant effects either on liver CSAD expression or taurine synthesis in male mice and suggests that the effects of sex steroids on taurine synthesis in the liver have gender differences. These new findings are the potential for establishing effective protective and therapeutic strategies for liver diseases.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Target of rapamycin signaling couples energy to oxygen sensing to modulate hypoxic gene expression in Arabidopsis.

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes.

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups. A-type PCOs are conserved across all plant species and are generally unaffected at the messenger RNA level by oxygen availability. Instead, B-type PCOs appeared in spermatophytes to acquire transcriptional regulation in response to hypoxia. The inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperms. Therefore, while it is possible to distinguish two clades within the PCO family, we conclude that they all contribute to restrain the anaerobic transcriptional programme in normoxic conditions and together generate a molecular switch to toggle the hypoxic response.

DFT insights into the mechanism of O2 activation catalyzed by a structural and functional model of cysteine dioxygenase with tris(2-pyridyl)methane-based ligand architecture.

Cysteine dioxygenation is an important step in the metabolism of toxic L-cysteine (Cys) in the human body, carried out by cysteine dioxygenase enzyme (CDO). The disruption of this process is found to elicit neurological health issues. This work reports a computational investigation of mechanistic aspects of this reaction, using a recently reported tris(2-pyridyl)methane-based biomimetic model complex of CDO. The computed results indicate that, the initial SO2 bond formation process is the slowest step in the S-dioxygenation process, possessing an activation barrier of 12.7 kcal/mol. The remaining steps were found to be downhill requiring very small activation energies. The transition states were found to undergo spin crossover between triplet and quintet states, while the singlet surface remained unstable throughout the entire reaction. In essence, the mechanistic scheme and multistate reactivity pattern together with the relatively small computed rate-limiting activation barrier as well as the exothermic formation energy demonstrate that the model complex is an efficient biomimetic CDO model. In addition, the study also substantiates the involvement of Fe(IV)oxido intermediates in the mechanism of S-dioxygenation by the chosen model complex. The insights derived from the O2 activation process might pave way for development of more accurate CDO model catalysts that might be capable of even more efficiently mimicking the geometric, spectroscopic and functional features of the CDO enzyme.

Assessing In Vivo Oxygen Dynamics Using Plant N-Terminal Degrons in Saccharomyces cerevisiae.

The expression of plant cysteine oxidase (PCO) enzyme in Saccharomyces cerevisiae enables the Arg/Cys N-degron pathway (Cys-NDP) for selective protein degradation that, in plants, functions as direct oxygen perception mechanism. A synthetic construct based on the plant Cys-NDP substrate related to apetala 2.12 (RAP2.12), the dual luciferase oxygen reporter (DLOR), exploits the N-terminal Cys of RAP2.12, and its oxygen-dependent degradation through the Cys-NDP. The luminescent output of DLOR can be used as a proxy for intracellular oxygen dynamics in budding yeast. Replacement of the luciferase reporter of the DLOR with fluorescent proteins would furthermore facilitate the imaging of reporter dynamics in living cells. In this chapter, we describe the methods for delivering the DLOR synthetic construct to yeast and calibrating its output by means of oxygen quantification in the culture with a physical oxygen sensor. We explain the setup needed to carry out hypoxic treatments with several colonies as replicates. We also describe the method to measure oxygen concentration in the culture, the closest indication of intracellular oxygen levels, as a way that would serve to calibrate the DLOR output. Finally, we propose a strategy to replace the luminescent reporters in the DLOR with fluorescent proteins to visualize oxygen dynamics in vivo.

CCl4 inhibits the expressions of hepatic taurine biosynthetic enzymes and taurine synthesis in the progression of mouse liver fibrosis.

Carbon tetrachloride (CCl4) is a widely used hepatotoxin for the studies of liver fibrosis and cirrhosis, and taurine has function to abate liver fibrosis induced by CCl4. But the interacting mechanisms between taurine and CCl4 in liver are still largely unknown. These made us to hypothesize that CCl4 may induce liver fibrosis by affecting the expressions of taurine biosynthetic enzymes and taurine synthesis. We thus assayed the expressions of hepatic cysteine dioxygenase (CDO), cysteine sulfonate acid decarboxylase (CSAD) and taurine transporter (TauT) in the progression of mouse liver fibrosis induced by CCl4. The results demonstrated that CCl4 treatment markedly decreased hepatic CSAD, CDO expressions, and taurine levels in hepatic tissue, although TauT expression did not exhibit significant decline. It was contrasting that hepatic α-SMA, serum AST, ALT, ALP kept increasing, which were accompanied by the pathological characters of liver, whereas taurine supplement attenuated the progression of liver fibrosis induced by CCl4. These results demonstrate that CCl4 may induce liver fibrosis by inhibiting hepatic CSAD and CDO expressions and taurine synthesis, which are crucial for our understanding the mechanisms of liver fibrosis induced by CCl4, and also potential for establishing therapeutic strategies of liver fibrosis and related diseases.