Key Branch Point Research Articles

Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine.

Through the shikimate pathway, a massive metabolic flux connects the central carbon metabolism with the synthesis of chorismate, the common precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as other compounds, including salicylate or folate. The alternative metabolic channeling of chorismate involves a key branch-point, finely regulated by aromatic amino acid levels. Chorismate mutase catalyzes the conversion of chorismate to prephenate, a precursor of phenylalanine and tyrosine and thus a vast repertoire of fundamental derived compounds, such as flavonoids or lignin. The regulation of this enzyme has been addressed in several plant species, but no study has included conifers or other gymnosperms, despite the importance of the phenolic metabolism for these plants in processes such as lignification and wood formation. Here, we show that maritime pine (Pinus pinaster Aiton) has two genes that encode for chorismate mutase, PpCM1 and PpCM2. Our investigations reveal that these genes encode plastidial isoenzymes displaying activities enhanced by tryptophan and repressed by phenylalanine and tyrosine. Using phylogenetic studies, we have provided new insights into the possible evolutionary origin of the cytosolic chorismate mutases in angiosperms involved in the synthesis of phenylalanine outside the plastid. Studies based on different platforms of gene expression and co-expression analysis have allowed us to propose that PpCM2 plays a central role in the phenylalanine synthesis pathway associated with lignification.

Natural variations in Crocus sativus lycopene epsilon cyclase (CstLcyE) alter carotenoid/apocarotenoid content and stress tolerance

Crocus sativus is an economically and pharmaceutically important crop. The dried stigmas of C. sativus flowers constitute saffron, the only commercial source of apocarotenoids like crocin, picrocrocin, and safranal. Some elite clones of C. sativus with higher crocin content were identified from plant populations over three years. Sequencing approach demonstrated that mutations are present in CstLycE. The C. sativus apocarotenoids are formed by cleavage of β-branch carotenoids. Cyclization of lycopene is the key branch point in carotenoid/apocarotenoid pathway. While lycopene β-cyclase converts lycopene into β-carotene, combined action of β and ε-cyclases forms α-carotene. Towards this, the mutant populations with critical mutations in CstLycE result in loss of function, and hence the metabolic flux was diverted towards β-branch. In-silico analysis and site-directed mutagenesis confirmed the critical nature of these mutations. To ascertain the role of CstLycE, transient over-expression and RNAi lines of CstLcyE in C. sativus were generated, and we observed enhanced β-branch carotenoid and apocarotenoid content in RNAi lines. The effect of CstLcyB and CstLcyE was compared, and CstLcyE RNAi lines had a more profound impact on apocarotenoid accumulation than CstLcyB over-expression lines. CstLcyE-RNAi plants also accumulated more abscisic acid and were more tolerant to abiotic stresses.

Carotenoid analysis and functional characterization of lycopene cyclases in Zinnia elegans L.

Carotenoids, a group of essential pigments for petal coloration in many species, could be generally categorized into linear and cyclic groups. The cyclization of the linear substrate lycopene at both end groups by β- and ε-cyclases (LCYBs and LCYEs, respectively) is a key branching point of the biosynthesis of cyclic carotenoids in flowers. Common zinnia (Zinnia elegans L.), an Asteraceae family plant that has been widely used in landscape greening, is best known for its brilliant flower colors. However, the mechanism of its petal coloration is not well studied, especially with respect to carotenoid pigmentation. In this study, total carotenoid content and carotenoid compositions of petals were analyzed in two common zinnia cultivars, 'Dreamland Red’ (DRE) and 'Dreamland Yellow’ (DY). Despite the lighter color of DY petals, the total carotenoid content in DY flowers was significantly higher than that in DRE. Metabolomic analysis revealed that the major carotenoids of both cultivars are cyclic carotenoids, with β-carotene being the most abundant. In addition, one LCYB, LCYE, and capsanthin/capsorubin synthase (CCS) gene were identified from Z. elegans by homology-based search in transcriptome. After cloning and sequencing, full-length open reading frame of each gene is exactly the same between two cultivars. Bacterial pigment complementation experiments revealed that ZeLCYB and ZeCCS can cyclize lycopene at both ends to produce β-carotene. Unlike most LCYEs characterized from higher plants, ZeLCYE produced predominantly ε-carotene in E. coli system. However, ZeLCYE is likely to have a lower affinity for lycopene or lower activity compared with ZeLCYB. Gene expression analysis showed that DY has higher expression levels of ZeLCYB and ZeCCS than DRE at specific developmental stages, consistent with its higher total carotenoid content. This work would lay a solid foundation for future studies on carotenoid accumulation in Z. elegans.

Metabolic Rewiring of Kynurenine Pathway during Hepatic Ischemia-Reperfusion Injury Exacerbates Liver Damage by Impairing NAD Homeostasis.

Hepatic ischemia-reperfusion (IR) injury remains a common issue lacking effective strategy and validated pharmacological targets. Here, using an unbiased metabolomics screen, this study finds that following murine hepatic IR, liver 3-hydroxyanthranilic acid (3-HAA) and quinolinic acid (QA) decline while kynurenine and kynurenic acid (KYNA) increase. Kynurenine aminotransferases 2, functioning at the key branching point of the kynurenine pathway (KP), is markedly upregulated in hepatocytes during ischemia, shifting the kynurenine metabolic route from 3-HAA and QA to KYNA synthesis. Defects in QA synthesis impair de novo nicotinamide adenine dinucleotide (NAD) biosynthesis, rendering the hepatocytes relying on the salvage pathway for maintenance of NAD and cellular antioxidant defense. Blocking the salvage pathway following IR by the nicotinamide phosphoribosyltransferase inhibitor FK866 exacerbates liver oxidative damage and enhanced IR susceptibility, which can be rescued by the lipid peroxidation inhibitor Liproxstatin-1. Notably, nicotinamide mononucleotide administration once following IR effectively boosts NAD and attenuated IR-induced oxidative stress, inflammation, and cell death in the murine model. Collectively, the findings reveal that metabolic rewiring of the KP partitions it away from NAD synthesis in hepatic IR pathophysiology, and provide proof of concept that NAD augmentation is a promising therapeutic measure for IR-induced liver injury.

Single-cell transcriptomics reveals the cell fate transitions of human dopaminergic progenitors derived from hESCs

BackgroundMidbrain dopaminergic (DA) progenitors derived from human pluripotent stem cells are considered to be a promising treatment for Parkinson’s disease (PD). However, the differentiation process produces undesired cell types, which influence the in vivo evaluation of DA cells. In this paper, we analyze the cell fate choice during differentiation and provide valuable information on cell preparation.MethodsHuman embryonic stem cells were differentiated into DA progenitors. We applied single-cell RNA sequencing (scRNA-seq) of the differentiation cells at different time points and investigated the gene expression profiles. Based on the differentially expressed genes between DA and non-DA cells, we investigated the impact of LGI1 (DA enriched) overexpression on DA differentiation and the enrichment effect of CD99 (non-DA enriched) sorting.ResultsTranscriptome analyses revealed the DA differentiation trajectory as well as non-DA populations and three key lineage branch points. Using genetic gain- and loss-of-function approaches, we found that overexpression of LGI1, which is specific to EN1+ early DA progenitors, can promote the generation of TH+ neurons. We also found that choroid plexus epithelial cells and DA progenitors are major components of the final product (day 25), and CD99 was a specific surface marker of choroid plexus epithelial cells. Sorting of CD99− cells eliminated major contaminant cells and improved the purity of DA progenitors.ConclusionsOur study provides the single-cell transcriptional landscape of in vitro DA differentiation, which can guide future improvements in DA preparation and quality control for PD cell therapy.

Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis.

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon.

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

Acid complexation of iron controls the fate of hydrogen peroxide in model wine

At a key branchpoint in wine oxidation, hydrogen peroxide reacts either with iron(II), leading to the Fenton oxidation of ethanol, or with sulfur dioxide, precluding oxidation. The fate of H2O2 was investigated in anoxic model wines with different pH and acid buffers. In the absence of SO2, anoxic conditions allowed the stoichiometric production of acetaldehyde from H2O2 despite iron(II) being limiting, indicating efficient iron redox cycling. Acetaldehyde production was faster at pH 4.0 than at pH 3.0, attributable largely to increased iron complexation. Citrate allowed the most rapid acetaldehyde formation, while the comparative effects of tartrate and malate were pH-dependent, likely due to differences in their iron-chelating abilities. The inclusion of SO2 greatly diminished acetaldehyde formation, but did not prevent it, and reduced the differential effects of pH and acid composition. Findings overall suggest management of wine acidity can significantly affect the rate and outcome of oxidation.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The gene encoding lycopene epsilon cyclase of celery enhanced lutein and β-carotene contents and confers increased salt tolerance in Arabidopsis

Celery (Apium graveolens L.) is a leafy vegetable of Apiaceae, which is greatly popular because of its rich nutrients. Lutein and β-carotene are two important carotenoids. Lycopene epsilon cyclase (LCY-ε) is a key branch point enzyme in the carotenoid biosynthetic pathway. In this study, we cloned the AgLCY-ε gene from celery and overexpressed it in Arabidopsis. The results showed that both lutein and β-carotene accumulation increased significantly in transgenic Arabidopsis hosting AgLCY-ε gene, compared with wild type (WT) plants. The transcription levels of AtPSY and AtCRTISO genes involved in carotenoids biosynthesis also increased in transgenic lines. One-month-old transgenic Arabidopsis seedlings were treated with 200 mM NaCl. The malondialdehyde (MDA) content in transgenic Arabidopsis plants after salt treatment was significantly lower, and the activities of the two antioxidant enzymes, superoxide dismutase (SOD) and peroxidase (POD), were significantly increased than that of WT plants. Overexpression of AgLCY-ε gene showed increased lutein and β-carotene accumulations, and enhanced salt tolerance in transgenic plants.

Parallel Single-Cell RNA-Seq and Genetic Recording Reveals Lineage Decisions in Developing Embryoid Bodies

SUMMARYEarly developmental specification can be modeled by differentiating embryonic stem cells (ESCs) to embryoid bodies (EBs), a heterogeneous mixture of three germ layers. Here, we combine single-cell transcriptomics and genetic recording to characterize EB differentiation. We map transcriptional states along a time course and model cell fate trajectories and branchpoints as cells progress to distinct germ layers. To validate this inferential model, we propose an innovative inducible genetic recording technique that leverages recombination to generate cell-specific, timestamp barcodes in a narrow temporal window. We validate trajectory architecture and key branchpoints, including early specification of a primordial germ cell (PGC)-like lineage from preimplantation epiblast-like cells. We further identify a temporally defined role of DNA methylation in this PGC-epiblast decision. Our study provides a high-resolution lineage map for an organoid model of embryogenesis, insights into epigenetic determinants of fate specification, and a strategy for lineage mapping of rapid differentiation processes.

The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway

The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3′H and F3′5′H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3′5′H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.

The Cholesterol Synthesis Enzyme Lanosterol 14α‐Demethylase is Post‐Translationally Regulated by the E3 Ubiquitin Ligase MARCH6

Cholesterol synthesis is a complex process with over twenty enzymes involved. Each enzyme within the pathway undergoes distinct regulation to maintain cholesterol homeostasis. Typically, enzymes within the pathway are degraded in response to a high sterol status, but some are highly stable. In this study, we demonstrate that an enzyme at the branch point in the pathway, lanosterol 14α‐demethylase (LDM), undergoes unique regulation. We show that the preceding enzyme, lanosterol synthase (LSS) is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols but by nitric oxide. Here we identify that the important E3 ubiquitin ligase, MARCH6, is responsible for facilitating the ubiquitination and proteasomal degradation of LDM and also the terminal cholesterol synthesis enzyme, 24‐dehydrocholesterol reductase (DHCR24). Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase which targets multiple steps in a single biochemical pathway and indicates new facets in the control of cholesterol synthesis.Support or Funding InformationThis work was supported by the Australian Research Council Grant DP170101178. The PhD students were funded by the Research Training Program from the Australian Government Department of Education.

The Terrestrial Migration of Vertebrates Drove the Evolution of ENaC Activation by Cleavage α

The epithelial Na+ channel (ENaC) is critical to regulating extracellular fluids through its activity in several epithelia, including the aldosterone‐sensitive distal nephron and colon, and the airway and alveoli. Human ENaC subunits are subject to activating proteolysis. The ENaC α and γ subunits may each be cleaved twice at polybasic tracts in their respective extracellular GRIP domains. This liberates imbedded inhibitory tracts, which activate the channel. Activation through either subunit requires double cleavage. To examine the evolution of ENaC cleavage sites, we generated a phylogenetic tree of ENaC and ENaC‐related subunits to identify key branch points in the evolution of ENaC subunits. The four ENaC subunits arose from three gene duplication events. First, a single gene diverged to an α/δ precursor and a β/γ precursor. Second, the β/γ precursor diverged to β and γ subunits. These two events occurred before the emergence of jawless fishes, as lampreys have α, β, and γ subunits. After the emergence of jawed fishes, α and δ diverged from the α/δ precursor; the lobe‐finned coelacanth is earliest rooted species to have all four subunits. We observed that the activating cleavage sites in the α and γ subunits appeared around the transition to terrestrial life. Among the fishes, only the Australian lungfish γ subunit had two cleavage sites, and all tetrapod α and γ subunits had two apparent cleavage sites. We ran nested likelihood models in BayesTraits and found a significant relationship between terrestrial status and having two cleavage sites in the α and γ subunits. Both sites showed a statistically significant coevolutionary pattern with the terrestrial state (proximal site, p = 0.004; distal site, p = 0.006). The sites also showed a coevolutionary pattern with each other (p = 0.0003). In contrast, the C‐terminal PY motif, which is important for aldosterone regulation through Sgk1/Nedd4‐2, did not show a dependence on terrestrial status (p = 0.58). We also performed ancestral reconstructions to determine whether these protein sequences arose through divergent or convergent evolution. Competing evolutionary models, where common ancestors were constrained to states reflecting each model, were compared using marginal likelihoods from Markov chain Monte Carlo simulations of each model. Our analysis strongly supports a convergent evolutionary model for the cleavage sites, and a divergent evolutionary model for the PY motifs. Our data suggest that aldosterone signaling adopted both long existing and newly evolved mechanisms to regulate ENaC function.

A metabolic switch regulates the transition between growth and diapause in C. elegans

BackgroundMetabolic activity alternates between high and low states during different stages of an organism’s life cycle. During the transition from growth to quiescence, a major metabolic shift often occurs from oxidative phosphorylation to glycolysis and gluconeogenesis. We use the entry of Caenorhabditis elegans into the dauer larval stage, a developmentally arrested stage formed in response to harsh environmental conditions, as a model to study the global metabolic changes and underlying molecular mechanisms associated with growth to quiescence transition.ResultsHere, we show that the metabolic switch involves the concerted activity of several regulatory pathways. Whereas the steroid hormone receptor DAF-12 controls dauer morphogenesis, the insulin pathway maintains low energy expenditure through DAF-16/FoxO, which also requires AAK-2/AMPKα. DAF-12 and AAK-2 separately promote a shift in the molar ratios between competing enzymes at two key branch points within the central carbon metabolic pathway diverting carbon atoms from the TCA cycle and directing them to gluconeogenesis. When both AAK-2 and DAF-12 are suppressed, the TCA cycle is active and the developmental arrest is bypassed.ConclusionsThe metabolic status of each developmental stage is defined by stoichiometric ratios within the constellation of metabolic enzymes driving metabolic flux and controls the transition between growth and quiescence.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.

Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.

Evolutionary Model of Plastidial RNA Editing in Angiosperms Presumed from Genome-Wide Analysis of Amborella trichopoda.

Amborella trichopoda is placed close to the base of the angiosperm lineage (basal angiosperm). By genome-wide RNA sequencing, we identified 184C-to-U RNA editing sites in the plastid genome of Amborella. This number is much higher than that observed in other angiosperms including maize (44 sites), rice (39 sites) and grape (115 sites). Despite the high frequency of RNA editing, the biased distribution of RNA editing sites in the genome, target codon preference and nucleotide preference adjacent to the edited cytidine are similar to that in other angiosperms, suggesting a common editing machinery. Consistent with this idea, the Amborella nuclear genome encodes 2-3 times more of the E- and DYW-subclass members of pentatricopeptide repeat proteins responsible for RNA editing site recognition in plant organelles. Among 165 editing sites in plastid protein coding sequences in Amborella, 100 sites were conserved at least in one out of 38 species selected to represent key branching points of the angiosperm phylogenetic tree. We assume these 100 sites represent at least a subset of the sites in the plastid editotype of ancestral angiosperms. We then mapped the loss and gain of editing sites on the phylogenetic tree of angiosperms. Our results support the idea that the evolution of angiosperms has led to the loss of RNA editing sites in plastids.

Branching points and transition pathways in the Greek Natural Gas Regime, 1966–2016

This paper studies the emerging natural gas transitions in Greece from 1966 to 2016. We place the actors and the governing processes in historical perspective, to position the regime actors, their power and their agency in the context of decision making that influenced the transition pathway and its key branching points. We argue that during the two transitions there was a passage from years of politicized visions promoted and dominated by national and local actors to years of contested visions of Greece as a transnational natural gas corridor, promoted both by national and transnational actors. There has been a reconfiguration of the governance logics action space because of the increasing agency and power of transnational actors (the EU and the ‘Troika’) especially at times of political or financial crisis. The paper shows the importance of considering how transitions are affected by asymmetries in power and agency between national and international actors and indeed between different actors in a state.

Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance.

This article develops a holistic view on production of reactive oxygen species (ROS) by 2-oxo acid dehydrogenase complexes. Recent Advances: Catalytic and structural properties of the complexes and their components evolved to minimize damaging effects of side reactions, including ROS generation, simultaneously exploiting the reactions for homeostatic signaling. Side reactions of the complexes, characterized in vitro, are analyzed in view of protein interactions and conditions in vivo. Quantitative data support prevalence of the forward 2-oxo acid oxidation over the backward NADH oxidation in feeding physiologically significant ROS production by the complexes. Special focus on interactions between the active sites within 2-oxo acid dehydrogenase complexes highlights the central relevance of the complex-bound thiyl radicals in regulation of and signaling by complex-generated ROS. The thiyl radicals arise when dihydrolipoyl residues of the complexes regenerate FADH2 from the flavin semiquinone coproduced with superoxide anion radical in 1e- oxidation of FADH2 by molecular oxygen. Interaction of 2-oxo acid dehydrogenase complexes with thioredoxins (TRXs), peroxiredoxins, and glutaredoxins mediates scavenging of the thiyl radicals and ROS generated by the complexes, underlying signaling of disproportional availability of 2-oxo acids, CoA, and NAD+ in key metabolic branch points through thiol/disulfide exchange and medically important hypoxia-inducible factor, mammalian target of rapamycin (mTOR), poly (ADP-ribose) polymerase, and sirtuins. High reactivity of the coproduced ROS and thiyl radicals to iron/sulfur clusters and nitric oxide, peroxynitrite reductase activity of peroxiredoxins and transnitrosylating function of thioredoxin, implicate the side reactions of 2-oxo acid dehydrogenase complexes in nitric oxide-dependent signaling and damage.