NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis.

Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine-tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow.

Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.

Understanding the Intersections between Metabolism and Cancer Biology.

Transformed cells adapt metabolism to support tumor initiation and progression. Specific metabolic activities can participate directly in the process of transformation or support the biological processes that enable tumor growth. Exploiting cancer metabolism for clinical benefit requires defining the pathways that are limiting for cancer progression and understanding the context specificity of metabolic preferences and liabilities in malignant cells. Progress toward answering these questions is providing new insight into cancer biology and can guide the more effective targeting of metabolism to help patients.

Co-evolution of Hormone Metabolism and Signaling Networks Expands Plant Adaptive Plasticity.

Classically, hormones elicit specific cellular responses by activating dedicated receptors. Nevertheless, the biosynthesis and turnover of many of these hormone molecules also produce chemically related metabolites. These molecules may also possess hormonal activities; therefore, one or more may contribute to the adaptive plasticity of signaling outcomes in host organisms. Here, we show that a catabolite of the plant hormone abscisic acid (ABA), namely phaseic acid (PA), likely emerged in seed plants as a signaling molecule that fine-tunes plant physiology, environmental adaptation, and development. This trait was facilitated by both the emergence-selection of a PA reductase that modulates PA concentrations and by the functional diversification of the ABA receptor family to perceive and respond to PA. Our results suggest that PA serves as a hormone in seed plants through activation of a subset of ABA receptors. This study demonstrates that the co-evolution of hormone metabolism and signaling networks can expand organismal resilience.

An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.

Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

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.

Phase separation of FSP1 promotes ferroptosis.

Ferroptosis is evolving as a highly promising approach to combat difficult-to-treat tumour entities including therapy-refractory and dedifferentiating cancers1-3. Recently, ferroptosis suppressor protein-1 (FSP1), along with extramitochondrial ubiquinone or exogenous vitamin K and NAD(P)H/H+ as an electron donor, has been identified as the second ferroptosis-suppressing system, which efficiently prevents lipid peroxidation independently of the cyst(e)ine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) axis4-6. To develop FSP1 inhibitors as next-generation therapeutic ferroptosis inducers, here we performed a small molecule library screen and identified the compound class of 3-phenylquinazolinones (represented by icFSP1) as potent FSP1 inhibitors. We show that icFSP1, unlike iFSP1, the first described on-target FSP1 inhibitor5, does not competitively inhibit FSP1 enzyme activity, but instead triggers subcellular relocalization of FSP1 from the membrane and FSP1 condensation before ferroptosis induction, in synergism with GPX4 inhibition. icFSP1-induced FSP1 condensates show droplet-like properties consistent with phase separation, an emerging and widespread mechanism to modulate biological activity7. N-terminal myristoylation, distinct amino acid residues and intrinsically disordered, low-complexity regions in FSP1 were identified to be essential for FSP1-dependent phase separation in cells and in vitro. We further demonstrate that icFSP1 impairs tumour growth and induces FSP1 condensates in tumours in vivo. Hence, our results suggest that icFSP1 exhibits a unique mechanism of action and synergizes with ferroptosis-inducing agents to potentiate the ferroptotic cell death response, thus providing a rationale for targeting FSP1-dependent phase separation as an efficient anti-cancer therapy.

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.

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.

Lysine acetylation restricts mutant IDH2 activity to optimize transformation in AML cells.

Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH). Mechanistically, K413 acetylation of mitochondrial mIDH2 is achieved through a series of hierarchical phosphorylation events mediated by tyrosine kinase FLT3, which phosphorylates mIDH2 to recruit upstream mitochondrial acetyltransferase ACAT1 and simultaneously activates ACAT1 and inhibits upstream mitochondrial deacetylase SIRT3 through tyrosine phosphorylation. Moreover, we found that the intrinsic enzyme activity of mIDH2 is much higher than mIDH1, thus the inhibitory K413 acetylation optimizes leukemogenic ability of mIDH2 in AML cells by both producing sufficient 2-HG for transformation and avoiding cytotoxic accumulation of intracellular 2-HG.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1.

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

A plant-derived natural photosynthetic system for improving cell anabolism.

Insufficient intracellular anabolism is a crucial factor involved in many pathological processes in the body1,2. The anabolism of intracellular substances requires the consumption of sufficient intracellular energy and the production of reducing equivalents. ATP acts as an 'energy currency' for biological processes in cells3,4, and the reduced form of NADPH is a key electron donor that provides reducing power for anabolism5. Under pathological conditions, it is difficult to correct impaired anabolism and to increase insufficient levels of ATP and NADPH to optimum concentrations1,4,6-8. Here we develop an independent and controllable nanosized plant-derived photosynthetic system based on nanothylakoid units (NTUs). To enable cross-species applications, we use a specific mature cell membrane (the chondrocyte membrane (CM)) for camouflage encapsulation. As proof of concept, we demonstrate that these CM-NTUs enter chondrocytes through membrane fusion, avoid lysosome degradation and achieve rapid penetration. Moreover, the CM-NTUs increase intracellular ATP and NADPH levels in situ following exposure to light and improve anabolism in degenerated chondrocytes. They can also systemically correct energy imbalance and restore cellular metabolism to improve cartilage homeostasis and protect against pathological progression of osteoarthritis. Our therapeutic strategy for degenerative diseases is based on a natural photosynthetic system that can controllably enhance cell anabolism by independently providing key energy and metabolic carriers. This study also provides an enhanced understanding of the preparation and application of bioorganisms and composite biomaterials for the treatment of disease.

Dynamic Regulation of ME1 Phosphorylation and Acetylation Affects Lipid Metabolism and Colorectal Tumorigenesis.

PGAM5 is a mitochondrial serine/threonine phosphatase that regulates multiple metabolic pathways and contributes to tumorigenesis in a poorly understood manner. We show here that PGAM5 inhibition attenuates lipid metabolism and colorectal tumorigenesis in mice. PGAM5-mediated dephosphorylation of malic enzyme 1 (ME1) at S336 allows increased ACAT1-mediated K337 acetylation, leading to ME1 dimerization and activation, both of which are reversed by NEK1 kinase-mediated S336 phosphorylation. SIRT6 deacetylase antagonizes ACAT1 function in a manner that involves mutually exclusive ME1 S336 phosphorylation and K337 acetylation. ME1 also promotes nicotinamide adenine dinucleotide phosphate (NADPH) production, lipogenesis, and colorectal cancers in which ME1 transcripts are upregulated and ME1 protein is hypophosphorylated at S336 and hyperacetylated at K337. PGAM5 and ME1 upregulation occur via direct transcriptional activation mediated by ß-catenin/TCF1. Thus, the balance between PGAM5-mediated dephosphorylation of ME1 S336 and ACAT1-mediated acetylation of K337 strongly influences NADPH generation, lipid metabolism, and the susceptibility to colorectal tumorigenesis.

TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes.

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

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.

Hydrogen isotope fractionation is controlled by CO2 in coccolithophore lipids.

Hydrogen isotope ratios (δ2H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2H/1H fractionation in algal lipids by systematically manipulating temperature, light, and CO2(aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica. We analyze the hydrogen isotope fractionation in alkenones (αalkenone), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the αalkenone with increasing CO2(aq) and confirm αalkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ2H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2H-richer intracellular water, increasing αalkenone. Higher chloroplast CO2(aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of αalkenone to CO2(aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO2 at the Rubisco site, but rather that chloroplast CO2 varies with external CO2(aq). The pervasive inverse correlation of αalkenone with CO2(aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, αalkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis.

Plasticity of the selectivity filter is essential for permeation in lysosomal TPC2 channels.

Two-pore channels are pathophysiologically important Na+- and Ca2+-permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P2, they are more Na+-selective than when gated by the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na+ permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na+ to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na+ over Ca2+. Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions.

NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.