Connections Between Metabolism and Epigenetics in Programming Cellular Differentiation.

Researchers are intensifying efforts to understand the mechanisms by which changes in metabolic states influence differentiation programs. An emerging objective is to define how fluctuations in metabolites influence the epigenetic states that contribute to differentiation programs. This is because metabolites such as S-adenosylmethionine, acetyl-CoA, α-ketoglutarate, 2-hydroxyglutarate, and butyrate are donors, substrates, cofactors, and antagonists for the activities of epigenetic-modifying complexes and for epigenetic modifications. We discuss this topic from the perspective of specialized CD4+ T cells as well as effector and memory T cell differentiation programs. We also highlight findings from embryonic stem cells that give mechanistic insight into how nutrients processed through pathways such as glycolysis, glutaminolysis, and one-carbon metabolism regulate metabolite levels to influence epigenetic events and discuss similar mechanistic principles in T cells. Finally, we highlight how dysregulated environments, such as the tumor microenvironment, might alter programming events.

A protein blueprint of the diatom CO2-fixing organelle.

Diatoms are central to the global carbon cycle. At the heart of diatom carbon fixation is an overlooked organelle called the pyrenoid, where concentrated CO2 is delivered to densely packed Rubisco. Diatom pyrenoids fix approximately one-fifth of global CO2, but the protein composition of this organelle is largely unknown. Using fluorescence protein tagging and affinity purification-mass spectrometry, we generate a high-confidence spatially defined protein-protein interaction network for the diatom pyrenoid. Within our pyrenoid interaction network are 10 proteins with previously unknown functions. We show that six of these form a shell that encapsulates the Rubisco matrix and is critical for pyrenoid structural integrity, shape, and function. Although not conserved at a sequence or structural level, the diatom pyrenoid shares some architectural similarities to prokaryotic carboxysomes. Collectively, our results support the convergent evolution of pyrenoids across the two main plastid lineages and uncover a major structural and functional component of global CO2 fixation.

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation.

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO2 fixation. Diatoms fix up to 20% of global CO2, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO2 is assimilated in the ocean, a critical ecosystem undergoing rapid change.

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Conversion of Escherichia coli to Generate All Biomass Carbon from CO2.

The living world is largely divided into autotrophs that convert CO2 into biomass and heterotrophs that consume organic compounds. In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO2 as their sole carbon source has so far remained an outstanding challenge. Here, we report the achievement of this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all its biomass carbon from CO2. Reducing power and energy, but not carbon, are supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling.

Mitochondrial One-Carbon Pathway Supports Cytosolic Folate Integrity in Cancer Cells.

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Multiomics analyses reveal a critical role of selenium in controlling T cell differentiation in Crohn's disease.

Inflammatory bowel disease (IBD) mainly includes Crohn's disease (CD) and ulcerative colitis (UC). Immune disorders play an essential role in the pathogenesis of these two IBDs, but the differences in the immune microenvironment of the colon and their underlying mechanisms remain poorly investigated. Here we examined the immunological features and metabolic microenvironment of untreated individuals with IBD by multiomics analyses. Modulation of CD-specific metabolites, particularly reduced selenium, can obviously shape type 1 T helper (Th1) cell differentiation, which is specifically enriched in CD. Selenium supplementation suppressed the symptoms and onset of CD and Th1 cell differentiation via selenoprotein W (SELW)-mediated cellular reactive oxygen species scavenging. SELW promoted purine salvage pathways and inhibited one-carbon metabolism by recruiting an E3 ubiquitin ligase, tripartite motif-containing protein 21, which controlled the stability of serine hydroxymethyltransferase 2. Our work highlights selenium as an essential regulator of T cell responses and potential therapeutic targets in CD.

Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

A systematic exploration of bacterial form I rubisco maximal carboxylation rates.

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.

Acetylation coordinates the crosstalk between carbon metabolism and ammonium assimilation in Salmonella enterica.

Enteric bacteria use up to 15% of their cellular energy for ammonium assimilation via glutamine synthetase (GS)/glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in response to varying ammonium availability. However, the sensory mechanisms for effective and appropriate coordination between carbon metabolism and ammonium assimilation have not been fully elucidated. Here, we report that in Salmonella enterica, carbon metabolism coordinates the activities of GS/GDH via functionally reversible protein lysine acetylation. Glucose promotes Pat acetyltransferase-mediated acetylation and activation of adenylylated GS. Simultaneously, glucose induces GDH acetylation to inactivate the enzyme by impeding its catalytic centre, which is reversed upon GDH deacetylation by deacetylase CobB. Molecular dynamics (MD) simulations indicate that adenylylation is required for acetylation-dependent activation of GS. We show that acetylation and deacetylation occur within minutes of "glucose shock" to promptly adapt to ammonium/carbon variation and finely balance glutamine/glutamate synthesis. Finally, in a mouse infection model, reduced S. enterica growth caused by the expression of adenylylation-mimetic GS is rescued by acetylation-mimicking mutations. Thus, glucose-driven acetylation integrates signals from ammonium assimilation and carbon metabolism to fine-tune bacterial growth control.

One-Carbon Metabolism Supports S-Adenosylmethionine and Histone Methylation to Drive Inflammatory Macrophages.

Activated macrophages adapt their metabolic pathways to drive the pro-inflammatory phenotype, but little is known about the biochemical underpinnings of this process. Here, we find that lipopolysaccharide (LPS) activates the pentose phosphate pathway, the serine synthesis pathway, and one-carbon metabolism, the synergism of which drives epigenetic reprogramming for interleukin-1ß (IL-1ß) expression. Glucose-derived ribose and one-carbon units fed by both glucose and serine metabolism are synergistically integrated into the methionine cycle through de novo ATP synthesis and fuel the generation of S-adenosylmethionine (SAM) during LPS-induced inflammation. Impairment of these metabolic pathways that feed SAM generation lead to anti-inflammatory outcomes, implicating SAM as an essential metabolite for inflammatory macrophages. Mechanistically, SAM generation maintains a relatively high SAM:S-adenosylhomocysteine ratio to support histone H3 lysine 36 trimethylation for IL-1ß production. We therefore identify a synergistic effect of glucose and amino acid metabolism on orchestrating SAM availability that is intimately linked to the chromatin state for inflammation.

Closing the geologic carbon cycle.

Estimates of sedimentary organic carbon burial fluxes based on inventory and isotope mass balance methods have been divergent. A new calculation of the isotope mass balance using a revised assessment of the inputs to the ocean-atmosphere system resolves the apparent discrepancy. Inputs include weathering of carbonate and old kerogen, geogenic methane oxidation, and volcanic and metamorphic degassing. Volcanic and metamorphic degassing comprise ≈23% of the total C input. Inputs from isotopically light OCpetro and CH4-geo drive the mean δ13C of the input to =-8.0 ± 1.9‰, notably lower than the commonly assumed volcanic degassing value. The isotope mass balance model yields a modern burial flux =15.9 ± 6.6 Tmol y-1. The impact of the mid-Miocene Climatic Optimum isotope anomaly is an integrated excess deposition ≈ 4.3 × 106 Tmol between 18 and 11 Ma, which is both longer and larger than estimates for the total degassing by the Columbia River Basalt eruptions, implying a complex carbon system response to large eruptive events. Monte Carlo evaluation finds that late Cenozoic net growth of the carbonate reservoir is very likely while net growth of the Corg reservoir is less certain but more likely than not. At present, subduction does not appear to keep up with net sedimentation and the overall masses of sedimentary carbonate and organic carbon are likely increasing. Growth in the sedimentary Corg reservoir implies oxidation of the surface environment and likely increases in atmospheric pO2.

Variable carbon isotope fractionation of photosynthetic communities over depth in an open-ocean euphotic zone.

Marine particulate organic carbon (POC) contributes to carbon export, food webs, and sediments, but uncertainties remain in its origins. Globally, variations in stable carbon isotope ratios (δ13C values) of POC between the upper and lower euphotic zones (LEZ) indicate either varying aspects of photosynthetic communities or degradative alteration of POC. During summertime in the subtropical north Atlantic Ocean, we find that δ13C values of the photosynthetic product phytol decreased by 6.3‰ and photosynthetic carbon isotope fractionation (εp) increased by 5.6‰ between the surface and the LEZ-variation as large as that found in the geologic record during major carbon cycle perturbations, but here reflecting vertical variation in δ13C values of photosynthetic communities. We find that simultaneous variations in light intensity and phytoplankton community composition over depth may be important factors not fully accounted for in common models of photosynthetic carbon isotope fractionation. Using additional isotopic and cell count data, we estimate that photosynthetic and non-photosynthetic material (heterotrophs or detritus) contribute relatively constant proportions of POC throughout the euphotic zone but are isotopically more distinct in the LEZ. As a result, the large vertical differences in εp result in significant, but smaller, differences in the δ13C values of total POC across the same depths (2.7‰). Vertical structuring of photosynthetic communities and export potential from the LEZ may vary across current and past ocean ecosystems; thus, LEZ photosynthesis may influence the exported and/or sedimentary δ13C values of both phytol and total organic carbon and affect interpretations of εp over geologic time.

Reconciling carbon quality with availability predicts temperature sensitivity of global soil carbon mineralization.

Soil organic carbon (SOC) mineralization is a key component of the global carbon cycle. Its temperature sensitivity Q10 (which is defined as the factor of change in mineralization with a 10 °C temperature increase) is crucial for understanding the carbon cycle-climate change feedback but remains uncertain. Here, we demonstrate the universal control of carbon quality-availability tradeoffs on Q10. When carbon availability is not limited, Q10 is controlled by carbon quality; otherwise, substrate availability controls Q10. A model driven by such quality-availability tradeoffs explains 97% of the spatiotemporal variability of Q10 in incubations of soils across the globe and predicts a global Q10 of 2.1 ± 0.4 (mean ± one SD) with higher Q10 in northern high-latitude regions. We further reveal that global Q10 is predominantly governed by the mineralization of high-quality carbon. The work provides a foundation for predicting SOC dynamics under climate and land use changes which may alter soil carbon quality and availability.

Southern Ocean drives multidecadal atmospheric CO2 rise during Heinrich Stadials.

The last glacial period was punctuated by cold intervals in the North Atlantic region that culminated in extensive iceberg discharge events. These cold intervals, known as Heinrich Stadials, are associated with abrupt climate shifts worldwide. Here, we present CO2 measurements from the West Antarctic Ice Sheet Divide ice core across Heinrich Stadials 2 to 5 at decadal-scale resolution. Our results reveal multi-decadal-scale jumps in atmospheric CO2 concentrations within each Heinrich Stadial. The largest magnitude of change (14.0 ± 0.8 ppm within 55 ± 10 y) occurred during Heinrich Stadial 4. Abrupt rises in atmospheric CO2 are concurrent with jumps in atmospheric CH4 and abrupt changes in the water isotopologs in multiple Antarctic ice cores, the latter of which suggest rapid warming of both Antarctica and Southern Ocean vapor source regions. The synchroneity of these rapid shifts points to wind-driven upwelling of relatively warm, carbon-rich waters in the Southern Ocean, likely linked to a poleward intensification of the Southern Hemisphere westerly winds. Using an isotope-enabled atmospheric circulation model, we show that observed changes in Antarctic water isotopologs can be explained by abrupt and widespread Southern Ocean warming. Our work presents evidence for a multi-decadal- to century-scale response of the Southern Ocean to changes in atmospheric circulation, demonstrating the potential for dynamic changes in Southern Ocean biogeochemistry and circulation on human timescales. Furthermore, it suggests that anthropogenic CO2 uptake in the Southern Ocean may weaken with poleward strengthening westerlies today and into the future.

Reducing the uncertainty in estimating soil microbial-derived carbon storage.

Soil organic carbon (SOC) is the largest carbon pool in terrestrial ecosystems and plays a crucial role in mitigating climate change and enhancing soil productivity. Microbial-derived carbon (MDC) is the main component of the persistent SOC pool. However, current formulas used to estimate the proportional contribution of MDC are plagued by uncertainties due to limited sample sizes and the neglect of bacterial group composition effects. Here, we compiled the comprehensive global dataset and employed machine learning approaches to refine our quantitative understanding of MDC contributions to total carbon storage. Our efforts resulted in a reduction in the relative standard errors in prevailing estimations by an average of 71% and minimized the effect of global variations in bacterial group compositions on estimating MDC. Our estimation indicates that MDC contributes approximately 758 Pg, representing approximately 40% of the global soil carbon stock. Our study updated the formulas of MDC estimation with improving the accuracy and preserving simplicity and practicality. Given the unique biochemistry and functioning of the MDC pool, our study has direct implications for modeling efforts and predicting the land-atmosphere carbon balance under current and future climate scenarios.