Plant Cysteine Oxidases Research Articles

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 invivo 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 invitro. 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.

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.

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 kineticstudies 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 kineticassays 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.

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.

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.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

The N-terminal cysteine is a dual sensor of oxygen and oxidative stress

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.

Targeting plant cysteine oxidase activity for improved submergence tolerance.

Plant cysteine oxidases (PCOs) are plant O2 -sensing enzymes. They catalyse the O2 -dependent step which initiates the proteasomal degradation of Group VII ethylene response transcription factors (ERF-VIIs) via the N-degron pathway. When submerged, plants experience a reduction in O2 availability; PCO activity therefore decreases and the consequent ERF-VII stabilisation leads to upregulation of hypoxia-responsive genes which enable adaptation to low O2 conditions. Resulting adaptations include entering an anaerobic quiescent state to maintain energy reserves and rapid growth to escape floodwater and allow O2 transport to submerged tissues. Stabilisation of ERF-VIIs has been linked to improved survival post-submergence in Arabidopsis, rice (Oryza sativa) and barley (Hordeum vulgare). Due to climate change and increasing flooding events, there is an interest in manipulating the PCO/ERF-VII interaction as a method of improving yields in flood-intolerant crops. An effective way of achieving this may be through PCO inhibition; however, complete ablation of PCO activity is detrimental to growth and phenotype, likely due to other PCO-mediated roles. Targeting PCOs will therefore require either temporary chemical inhibition or careful engineering of the enzyme structure to manipulate their O2 sensitivity and/or substrate specificity. Sufficient PCO structural and functional information should make this possible, given the potential to engineer site-directed mutagenesis in vivo using CRISPR-mediated base editing. Here, we discuss the knowledge still required for rational manipulation of PCOs to achieve ERF-VII stabilisation without a yield penalty. We also take inspiration from the biocatalysis field to consider how enzyme engineering could be accelerated as a wider strategy to improve plant stress tolerance and productivity.

Emerging roles for thiol dioxygenases as oxygen sensors.

Cysteine dioxygenases, 3-mercaptopropionate dioxygenases and mercaptosuccinate dioxygenases are all thiol dioxygenases (TDOs) that catalyse oxidation of thiol molecules to sulphinates. They are Fe(II)-dependent dioxygenases with a cupin fold that supports a 3xHis metal-coordinating triad at the active site. They also have other, broadly common features including arginine residues involved in substrate carboxylate binding and a conserved trio of residues at the active site featuring a tyrosine important in substrate binding catalysis. Recently, N-terminal cysteinyl dioxygenase enzymes (NCOs) have been identified in plants (plant cysteine oxidases, PCOs), while human 2-aminoethanethiol dioxygenase (ADO) has been shown to act as both an NCO and a small molecule TDO. Although the cupin fold and 3xHis Fe(II)-binding triad seen in the small molecule TDOs are conserved in NCOs, other active site features and aspects of the overall protein architecture are quite different. Furthermore, the PCOs and ADO appear to act as biological O2 sensors, as shown by kinetic analyses and hypoxic regulation of the stability of their biological targets (N-terminal cysteine oxidation triggers protein degradation via the N-degron pathway). Here, we discuss the emergence of these two subclasses of TDO including structural features that could dictate their ability to bind small molecule or polypeptide substrates. These structural features may also underpin the O2 -sensing capability of the NCOs. Understanding how these enzymes interact with their substrates, including O2 , could reveal strategies to manipulate their activity, relevant to hypoxic disease states and plant adaptive responses to flooding.

Molecular basis for cysteine oxidation by plant cysteine oxidases from Arabidopsis thaliana

Plant Cysteine Oxidases (PCOs) play important roles in controlling the stability of Group VII ethylene response factors (ERF-VIIs) via Arg/N-degron pathway through catalyzing the oxidation of their N-Cys for subsequent Arginyl-tRNA--protein transferase 1 (ATE1) mediated arginine installation. Here we presented the crystal structures of PCO2, PCO4, and PCO5 from Arabidopsis thaliana (AtPCOs) and examined their in vitro activity by Mass spectrometry (MS). On the basis of Tris-bound AtPCO2, we modelled the structure of Cys-bound AtPCO2 and identified key AtPCO2 residues involved in N-Cys recognition and oxidation. Alanine substitution of potential N-Cys interaction residues impaired the activity of AtPCO5 remarkably. The structural research, complemented by mutagenesis and MS experiments, not only uncovers the substrate recognition and catalytic mode by AtPCOs, but also sheds light on the future design of potent inhibitors for plant cysteine oxidases.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

Oxygen sensing across kingdoms

Biochemistry The ability to sense and respond to changes in oxygen levels is critical for most forms of life. To date, mechanistic studies of this process in mammals have focused on the oxygen-sensitive stability of a transcription factor called hypoxia-inducible factor. Masson et al. discovered an enzymatic oxygen sensor in humans that is functionally identical to plant cysteine oxidases, enzymes that control responses to hypoxia in plants. The human and plant enzymes convert the N-terminal cysteine in substrate proteins to cysteine sulfinic acid, a modification that ultimately targets the proteins for degradation. Oxygen sensing is impaired in many human diseases, and further study of the human enzyme could help in the development of strategies for therapeutic intervention. Science , this issue p. [65][1] [1]: /lookup/doi/10.1126/science.aaw0112

Zinc Excess Induces a Hypoxia-Like Response by Inhibiting Cysteine Oxidases in Poplar Roots.

Poplar (Populus spp.) is a tree species considered for the remediation of soil contaminated by metals, including zinc (Zn). To improve poplar's capacity for Zn assimilation and compartmentalization, it is necessary to understand the physiological and biochemical mechanisms that enable these features as well as their regulation at the molecular level. We observed that the molecular response of poplar roots to Zn excess overlapped with that activated by hypoxia. Therefore, we tested the effect of Zn excess on hypoxia-sensing components and investigated the consequence of root hypoxia on poplar fitness and Zn accumulation capacity. Our results suggest that high intracellular Zn concentrations mimic iron deficiency and inhibit the activity of the oxygen sensors Plant Cysteine Oxidases, leading to the stabilization and activation of ERF-VII transcription factors, which are key regulators of the molecular response to hypoxia. Remarkably, excess Zn and waterlogging similarly decreased poplar growth and development. Simultaneous excess Zn and waterlogging did not exacerbate these parameters, although Zn uptake was limited. This study unveils the contribution of the oxygen-sensing machinery to the Zn excess response in poplar, which may be exploited to improve Zn tolerance and increase Zn accumulation capacity in plants.

The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors

Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors. However, their ability to respond to fluctuations in O2 concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1–5 to ascertain whether their activities are sensitive to O2 levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had Km(O2)app values in a physiologically relevant range, which should enable them to sensitively react to changes in O2 availability. This work validates an O2-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.

Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway

In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.