A Non-Functional Carbon Dioxide-Mediated Post-Translational Modification on Nucleoside Diphosphate Kinase of Arabidopsis thaliana.

The carbamate post-translational modification (PTM), formed by the nucleophilic attack of carbon dioxide by a dissociated lysine epsilon-amino group, is proposed as a widespread mechanism for sensing this biologically important bioactive gas. Here, we demonstrate the discovery and in vitro characterization of a carbamate PTM on K9 of Arabidopsis nucleoside diphosphate kinase (AtNDK1). We demonstrate that altered side chain reactivity at K9 is deleterious for AtNDK1 structure and catalytic function, but that CO2 does not impact catalysis. We show that nucleotide substrate removes CO2 from AtNDK1, and the carbamate PTM is functionless within the detection limits of our experiments. The AtNDK1 K9 PTM is the first demonstration of a functionless carbamate. In light of this finding, we speculate that non-functionality is a possible feature of the many newly identified carbamate PTMs.

Near atmospheric carbon dioxide activates plant ubiquitin cross-linking.

Background Identifying CO2-binding proteins is vital for our knowledge of CO2-regulated molecular processes. The carbamate post-translational modification is a reversible CO2-mediated adduct that can form on neutral N-terminal α-amino or lysine ε-amino groups. Methods We have developed triethyloxonium ion (TEO) as a chemical proteomics tool to trap the carbamate post-translational modification on protein covalently. We use 13C-NMR and TEO and identify ubiquitin as a plant CO2-binding protein. Results We observe the carbamate post-translational modification on the Arabidopsis thaliana ubiquitin ε-amino groups of lysines 6, 33, and 48. We show that biologically relevant near atmospheric PCO2 levels increase ubiquitin conjugation dependent on lysine 6. We further demonstrate that CO2 increases the ubiquitin E2 ligase (AtUBC5) charging step via the transthioesterification reaction in which Ub is transferred from the E1 ligase active site to the E2 active site. Conclusions and general significance Therefore, plant ubiquitin is a CO2-binding protein, and the carbamate post-translational modification represents a potential mechanism through which plant cells can respond to fluctuating PCO2.

Allophycocyanin A is a carbon dioxide receptor in the cyanobacterial phycobilisome.

Light harvesting is fundamental for production of ATP and reducing equivalents for CO2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO2. Here, we show that CO2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation.

Carbon Dioxide and the Carbamate Post-Translational Modification.

Carbon dioxide is essential for life. It is at the beginning of every life process as a substrate of photosynthesis. It is at the end of every life process as the product of post-mortem decay. Therefore, it is not surprising that this gas regulates such diverse processes as cellular chemical reactions, transport, maintenance of the cellular environment, and behaviour. Carbon dioxide is a strategically important research target relevant to crop responses to environmental change, insect vector-borne disease and public health. However, we know little of carbon dioxide's direct interactions with the cell. The carbamate post-translational modification, mediated by the nucleophilic attack by carbon dioxide on N-terminal α-amino groups or the lysine ɛ-amino groups, is one mechanism by which carbon dioxide might alter protein function to form part of a sensing and signalling mechanism. We detail known protein carbamates, including the history of their discovery. Further, we describe recent studies on new techniques to isolate this problematic post-translational modification.

Erratum: Physics of Brains.

[This corrects the article DOI: 10.1016/j.isci.2021.102877.].

Applying TADF Emitters in Bioimaging and Sensing-A Novel Approach Using Liposomes for Encapsulation and Cellular Uptake.

A new method for facilitating the delivery, uptake and intracellular localisation of thermally activated delayed fluorescence (TADF) complexes was developed. First, confinement of TADF complexes in liposomes was demonstrated, which were subsequently used as the delivery vehicle for cellular uptake. Confocal fluorescence microscopy showed TADF complexes subsequently localise in the cytoplasm of HepG2 cells. The procedures developed in this work included the removal of molecular oxygen in the liposome preparation without disrupting the liposome structures. Time-resolved fluorescence microscopy (point scanning) showed initial prompt fluorescence followed by a weak, but detectable, delayed fluorescence component for liposomal TADF internalised in HepG2 cells. By demonstrating that it is possible to deliver un-functionalised and/or unshielded TADF complexes, a sensing function for TADFs, such as molecular oxygen, can be envisaged.

Ubiquitin is a carbon dioxide-binding protein.

The identification of CO2-binding proteins is crucial to understanding CO2-regulated molecular processes. CO2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal α-amino or lysine ε-amino groups. We have previously developed triethyloxonium (TEO) ion as a chemical proteomics tool for covalent trapping of carbamates, and here, we deploy TEO to identify ubiquitin as a mammalian CO2-binding protein. We use 13C-NMR spectroscopy to demonstrate that CO2 forms carbamates on the ubiquitin N terminus and ε-amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-κB pathway activation. Our results show that ubiquitin is a CO2-binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO2.

A methodology for carbamate post-translational modification discovery and its application in Escherichia coli.

Carbon dioxide can influence cell phenotypes through the modulation of signalling pathways. CO2 regulates cellular processes as diverse as metabolism, cellular homeostasis, chemosensing and pathogenesis. This diversity of regulated processes suggests a broadly conserved mechanism for CO2 interactions with diverse cellular targets. CO2 is generally unreactive but can interact with neutral amines on protein under normal intracellular conditions to form a carbamate post-translational modification (PTM). We have previously demonstrated the presence of this PTM in a subset of protein produced by the model plant species Arabidopsis thaliana. Here, we describe a detailed methodology for identifying new carbamate PTMs in an extracted soluble proteome under biologically relevant conditions. We apply this methodology to the soluble proteome of the model prokaryote Escherichia coli and identify new carbamate PTMs. The application of this methodology, therefore, supports the hypothesis that the carbamate PTM is both more widespread in biology than previously suspected and may represent a broadly relevant mechanism for CO2-protein interactions.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

KEY POINTS: A moderate increase in PCO2 (55 mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. ABSTRACT: Cx26 hemichannels open in response to moderate elevations of CO2 ( PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30 mm propionate, we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

A DNA-Binding Bromodomain-Containing Protein Interacts with and Reduces Rx1-Mediated Immune Response to Potato Virus X.

Plant NLR proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming. Some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato binds and distorts double-stranded DNA. However, the components of the chromatin-localized Rx1 complex are largely unknown. Here, we report a physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP accumulates in the nucleoplasm and nucleolus, interacts with chromatin, and redistributes Rx1 to the nucleolus in a subpopulation of imaged cells. Rx1 overexpression reduces the interaction between NbDBCP and chromatin. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X (PVX), and this activity requires an intact bromodomain. Previously, Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor, NbGlk1. Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding, suggesting a mode of action for NbDBCP's inhibitory effect on immunity. This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception.

Author Correction: The identification of carbon dioxide mediated protein post-translational modifications.

The original version of this Article omitted the following from the Acknowledgements: 'This work was support by EPSRC grant EP/K504336/1 and Leverhulme Trust grant RPG-2016-017.' This has been corrected in both the PDF and HTML versions of the Article.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

The identification of carbon dioxide mediated protein post-translational modifications.

Carbon dioxide is vital to the chemistry of life processes including metabolism, cellular homoeostasis, and pathogenesis. CO2 is generally unreactive but can combine with neutral amines to form carbamates on proteins under physiological conditions. The most widely known examples of this are CO2 regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase and haemoglobin. However, the systematic identification of CO2-binding sites on proteins formed through carbamylation has not been possible due to the ready reversibility of carbamate formation. Here we demonstrate a methodology to identify protein carbamates using triethyloxonium tetrafluoroborate to covalently trap CO2, allowing for downstream proteomic analysis. This report describes the systematic identification of carbamates in a physiologically relevant environment. We demonstrate the identification of carbamylated proteins and the general principle that CO2 can impact protein biochemistry through carbamate formation. The ability to identify protein carbamates will significantly advance our understanding of cellular CO2 interactions.

The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and NbGlk1, a Golden2-like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions.

Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator-dependent anion and fluid secretion in airway epithelia.

Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine- and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2 -induced reduction in anion secretion was not due to a decrease in HCO3 (-) transport given that neither a change in CFTR-dependent HCO3 (-) efflux nor Na(+) /HCO3 (-) cotransporter-dependent HCO3 (-) influx were CO2 -sensitive. Hypercapnia also reduced the volume of forskolin-stimulated fluid secretion over 24 h, yet had no effect on the HCO3 (-) content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR-dependent, electrogenic Cl(-) and fluid secretion, but not CFTR-dependent HCO3 (-) secretion, which highlights a differential sensitivity of Cl(-) and HCO3 (-) transporters to raised CO2 in Calu-3 cells. Hypercapnia also reduced forskolin-stimulated CFTR-dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

Dynamic Transmission of Protein Allostery without Structural Change: Spatial Pathways or Global Modes?

We examine the contrast between mechanisms for allosteric signaling that involve structural change, and those that do not, from the perspective of allosteric pathways. In particular we treat in detail the case of fluctuation-allostery by which amplitude modulation of the thermal fluctuations of the elastic normal modes conveys the allosteric signal, and address the question of what an allosteric pathway means in this case. We find that a perturbation theory of thermal elastic solids and nonperturbative approach (by super-coarse-graining elasticity into internal bending modes) have opposite signatures in their structure of correlated pathways. We illustrate the effect from analysis of previous results from GlxR of Corynebacterium glutamicum, an example of the CRP/FNR transcription family of allosteric homodimers. We find that the visibility of both correlated pathways and disconnected sites of correlated motion in this protein suggests that mechanisms of local elastic stretch and bend are recruited for the purpose of creating and controlling allosteric cooperativity.

Ecto-5'-Nucleotidase, Adenosine and Transmembrane Adenylyl Cyclase Signalling Regulate Basal Carotid Body Chemoafferent Outflow and Establish the Sensitivity to Hypercapnia.

Carotid body (CB) stimulation by hypercapnia causes a reflex increase in ventilation and, along with the central chemoreceptors, this prevents a potentially lethal systemic acidosis. Control over the CB chemoafferent output during normocapnia and hypercapnia most likely involves multiple neurotransmitters and neuromodulators including ATP, acetylcholine, dopamine, serotonin and adenosine, but the precise role of each is yet to be fully established. In the present study, recordings of chemoafferent discharge frequency were made from the isolated in vitro CB in order to determine the contribution of adenosine, derived specifically from extracellular catabolism of ATP, in mediating basal chemoafferent activity and responses to hypercapnia. Pharmacological inhibition of ecto-5'-nucleotidase (CD73), a key enzyme required for extracellular generation of adenosine from ATP, using α,ß-methylene ADP, virtually abolished the basal normocapnic single fibre discharge frequency (superfusate PO(2) ~ 300 mmHg, PCO(2) ~ 40 mmHg) and diminished the chemoafferent response to hypercapnia (PCO(2) ~ 80 mmHg). These effects were mimicked by the blockade of adenosine receptors with 8-(p-sulfophenyl) theophylline. The excitatory impact of adenosinergic signalling on CB hypercapnic sensitivity is most likely to be conferred through changes in cAMP. Here, inhibition of transmembrane, but not soluble adenylate cyclases, reduced normocapnic single fibre activity and inhibited the elevation evoked by hypercapnia by approximately 50 %. These data therefore identify a functional role for CD73 derived adenosine and transmembrane adenylate cyclases, in modulating the basal chemoafferent discharge frequency and in priming the CB to hypercapnic stimulation.

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.