Selective activation of PFKL suppresses the phagocytic oxidative burst.

In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.

A White-Box Machine Learning Approach for Revealing Antibiotic Mechanisms of Action.

Current machine learning techniques enable robust association of biological signals with measured phenotypes, but these approaches are incapable of identifying causal relationships. Here, we develop an integrated "white-box" biochemical screening, network modeling, and machine learning approach for revealing causal mechanisms and apply this approach to understanding antibiotic efficacy. We counter-screen diverse metabolites against bactericidal antibiotics in Escherichia coli and simulate their corresponding metabolic states using a genome-scale metabolic network model. Regression of the measured screening data on model simulations reveals that purine biosynthesis participates in antibiotic lethality, which we validate experimentally. We show that antibiotic-induced adenine limitation increases ATP demand, which elevates central carbon metabolism activity and oxygen consumption, enhancing the killing effects of antibiotics. This work demonstrates how prospective network modeling can couple with machine learning to identify complex causal mechanisms underlying drug efficacy.

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.

NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells.

Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Awakening adult neural stem cells: NOX signalling as a positive regulator of the quiescence-to-proliferation transition in the Xenopus retina.

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

Immunity to pathogenic fungi in the eye.

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production.

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Lutetium texaphyrin: A photocatalyst that triggers pyroptosis via biomolecular photoredox catalysis.

Photon-controlled pyroptosis activation (PhotoPyro) is a promising technique for cancer immunotherapy due to its noninvasive nature, precise control, and ease of operation. Here, we report that biomolecular photoredox catalysis in cells might be an important mechanism underlying PhotoPyro. Our findings reveal that the photocatalyst lutetium texaphyrin (MLu) facilitates rapid and direct photoredox oxidation of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and various amino acids, thereby triggering pyroptosis through the caspase 3/GSDME pathway. This mechanism is distinct from the well-established role of MLu as a photodynamic therapy sensitizer in cells. Two analogs of MLu, bearing different coordinated central metal cations, were also explored as controls. The first control, gadolinium texaphyrin (MGd), is a weak photocatalyst but generates reactive oxygen species (ROS) efficiently. The second control, manganese texaphyrin (MMn), is ineffective as both a photocatalyst and a ROS generator. Neither MGd nor MMn was found to trigger pyroptosis under the conditions where MLu was active. Even in the presence of a ROS scavenger, treating MDA-MB-231 cells with MLu at concentrations as low as 50 nM still allows for pyroptosis photo-activation. The present findings highlight how biomolecular photoredox catalysis could contribute to pyroptosis activation by mechanisms largely independent of ROS.

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis.

The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.

A paREDOX in the control of cholesterol biosynthesis: Does the NADPH sensor and E3 ubiquitin ligase MARCHF6 protect mammalian cells during oxidative stress by controlling sterol biosynthesis?

Sterols and the reductant nicotinamide adenine dinucleotide phosphate (NADPH), essential for eukaryotic life, arose because of, and as an adaptation to, rising levels of molecular oxygen (O2). Hence, the NADPH and O2-intensive process of sterol biosynthesis is inextricably linked to redox status. In mammals, cholesterol biosynthesis is exquisitely regulated post-translationally by multiple E3 ubiquitin ligases, with membrane associated Really Interesting New Gene (RING) C3HC4 finger 6 (MARCHF6) degrading at least six enzymes in the pathway. Intriguingly, all these MARCHF6-dependent enzymes require NADPH. Moreover, MARCHF6 is activated by NADPH, although what this means for control of cholesterol synthesis is unclear. Indeed, this presents a paradox for how NADPH regulates this vital pathway, since NADPH is a cofactor in cholesterol biosynthesis and yet, low levels of NADPH should spare cholesterol biosynthesis enzymes targeted by MARCHF6 by reducing its activity. We speculate MARCHF6 helps mammalian cells adapt to oxidative stress (signified by low NADPH levels) by reducing degradation of cholesterogenic enzymes, thereby maintaining synthesis of protective cholesterol.

NADP(H)-dependent biocatalysis without adding NADP(H).

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP+/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP+ reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo-the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 102 to 103-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the "gain of function" reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.

ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

The tricarboxylic acid (TCA) cycle: a malleable metabolic network to counter cellular stress.

The tricarboxylic acid (TCA) cycle is a primordial metabolic pathway that is conserved from bacteria to humans. Although this network is often viewed primarily as an energy producing engine fueling ATP synthesis via oxidative phosphorylation, mounting evidence reveals that this metabolic hub orchestrates a wide variety of pivotal biological processes. It plays an important part in combatting cellular stress by modulating NADH/NADPH homeostasis, scavenging ROS (reactive oxygen species), producing ATP by substrate-level phosphorylation, signaling and supplying metabolites to quell a range of cellular disruptions. This review elaborates on how the reprogramming of this network prompted by such abiotic stress as metal toxicity, oxidative tension, nutrient challenge and antibiotic insult is critical for countering these conditions in mostly microbial systems. The cross-talk between the stressors and the participants of TCA cycle that results in changes in metabolite and nucleotide concentrations aimed at combatting the abiotic challenge is presented. The fine-tuning of metabolites mediated by disparate enzymes associated with this metabolic hub is discussed. The modulation of enzymatic activities aimed at generating metabolic moieties dedicated to respond to the cellular perturbation is explained. This ancient metabolic network has to be recognized for its ability to execute a plethora of physiological functions beyond its well-established traditional roles.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

NADPH oxidase-dependent H2O2 production mediates salicylic acid-induced salt tolerance in mangrove plant Kandelia obovata by regulating Na+/K+ and redox homeostasis.

Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.

Extracellular and intracellular destruction of Pseudomonas aeruginosa by Dictyostelium discoideum phagocytes mobilize different antibacterial mechanisms.

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.