Mitochondrial Metabolism Regulation of T Cell-Mediated Immunity.

Recent evidence supports the notion that mitochondrial metabolism is necessary for T cell activation, proliferation, and function. Mitochondrial metabolism supports T cell anabolism by providing key metabolites for macromolecule synthesis and generating metabolites for T cell function. In this review, we focus on how mitochondrial metabolism controls conventional and regulatory T cell fates and function.

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.

Ketolysis drives CD8+ T cell effector function through effects on histone acetylation.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

Transcription factors TEAD2 and E2A globally repress acetyl-CoA synthesis to promote tumorigenesis.

Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.

Coenzyme A precursors flow from mother to zygote and from microbiome to host.

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Mechanistic insight into allosteric activation of human pyruvate carboxylase by acetyl-CoA.

Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.

Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.

Stearate-derived very long-chain fatty acids are indispensable to tumor growth.

Reprogramming of lipid metabolism is emerging as a hallmark of cancer, yet involvement of specific fatty acids (FA) species and related enzymes in tumorigenesis remains unclear. While previous studies have focused on involvement of long-chain fatty acids (LCFAs) including palmitate in cancer, little attention has been paid to the role of very long-chain fatty acids (VLCFAs). Here, we show that depletion of acetyl-CoA carboxylase (ACC1), a critical enzyme involved in the biosynthesis of fatty acids, inhibits both de novo synthesis and elongation of VLCFAs in human cancer cells. ACC1 depletion markedly reduces cellular VLCFA but only marginally influences LCFA levels, including palmitate that can be nutritionally available. Therefore, tumor growth is specifically susceptible to regulation of VLCFAs. We further demonstrate that VLCFA deficiency results in a significant decrease in ceramides as well as downstream glucosylceramides and sphingomyelins, which impairs mitochondrial morphology and renders cancer cells sensitive to oxidative stress and cell death. Taken together, our study highlights that VLCFAs are selectively required for cancer cell survival and reveals a potential strategy to suppress tumor growth.

Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Activator of one protease transforms into inhibitor of another in response to nutritional signals.

All cells use proteases to adjust protein amounts. Proteases maintain protein homeostasis by degrading nonfunctional toxic proteins and play regulatory roles by targeting particular substrates in response to specific signals. Here we address how cells tune protease specificity to nutritional signals. We report that Salmonella enterica increases the specificity of the broadly conserved proteases Lon and ClpSAP by transforming the Lon activator and substrate HspQ into an inhibitor of the N-degron recognin ClpS, the adaptor of the ClpAP protease. We establish that upon acetylation, HspQ stops being a Lon activator and substrate and that the accumulated HspQ binds to ClpS, hindering degradation of ClpSAP substrates. Growth on glucose promotes HspQ acetylation by increasing acetyl-CoA amounts, thereby linking metabolism to proteolysis. By altering protease specificities but continuing to degrade junk proteins, cells modify the abundance of particular proteins while preserving the quality of their proteomes. This rapid response mechanism linking protease specificity to nutritional signals is broadly conserved.

ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly.

The electron transport chain (ETC) is an important participant in cellular energy conversion, but its biogenesis presents the cell with numerous challenges. To address these complexities, the cell utilizes ETC assembly factors, which include the LYR protein family. Each member of this family interacts with the mitochondrial acyl carrier protein (ACP), the scaffold protein upon which the mitochondrial fatty acid synthesis (mtFAS) pathway builds fatty acyl chains from acetyl-CoA. We demonstrate that the acylated form of ACP is an acetyl-CoA-dependent allosteric activator of the LYR protein family used to stimulate ETC biogenesis. By tuning ETC assembly to the abundance of acetyl-CoA, which is the major fuel of the TCA cycle and ETC, this system could provide an elegant mechanism for coordinating the assembly of ETC complexes with one another and with substrate availability.

The mitochondrion of Plasmodium falciparum is required for cellular acetyl-CoA metabolism and protein acetylation.

Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the apicoplast and the cytosol of malaria parasites, mitochondrial acetyl-CoA production is less well understood. In the current study, we performed metabolite-labeling experiments to measure endogenous metabolites in Plasmodium falciparum lines with genetic deletions affecting mitochondrial dehydrogenase activity. Our results show that the mitochondrion is required for cellular acetyl-CoA biosynthesis and identify a synthetic lethal relationship between the two main ketoacid dehydrogenase enzymes. The activity of these enzymes is dependent on the lipoate attachment enzyme LipL2, which is essential for parasite survival solely based on its role in supporting acetyl-CoA metabolism. We also find that acetyl-CoA produced in the mitochondrion is essential for the acetylation of histones and other proteins outside of the mitochondrion. Taken together, our results demonstrate that the mitochondrion is required for cellular acetyl-CoA metabolism and protein acetylation essential for parasite survival.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Acetyl-CoA-directed gene transcription in cancer cells.

Fluctuations in acetyl-coenzyme A (acetyl-CoA) levels have been previously associated with changes in global histone acetylation and gene expression. The study by Lee and colleagues (pp. 497-511) in this issue of Genes & Development demonstrates that acetyl-CoA can promote the up-regulation of cell migration- and adhesion-related genes in glioblastoma by controlling Ca2+-NFAT (nuclear factor of activated T cells) signaling.

A single Leishmania adenylate forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA.

Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.

Reciprocal regulation of cardiac ß-oxidation and pyruvate dehydrogenase by insulin.

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.

Sexually dimorphic acetyl-CoA biosynthesis and utilization in response to drought and exogenous acetic acid.

Female willows exhibit greater drought tolerance and benefit more from exogenous acetic acid (AA)-improved drought tolerance than males. However, the potential mechanisms driving these sex-specific responses remain unclear. To comprehensively investigate the sexually dimorphic responsive mechanisms of willows to drought and exogenous AA, here, we performed physiological, proteomic, Lys-acetylproteomic, and transgenic analyses in female and male Salix myrtillacea exposed to drought and AA-applicated drought treatments, focusing on protein abundance and lysine acetylation (LysAc) changes. Drought-tolerant females suffered less drought-induced photosynthetic and oxidative damage, did not activate AA and acetyl-CoA biosynthesis, TCA cycle, fatty acid metabolism, and jasmonic acid signaling as strongly as drought-sensitive males. Exogenous AA caused overaccumulation of endogenous AA and inhibition of acetyl-CoA biosynthesis and utilization in males. However, exogenous AA greatly enhanced acetyl-CoA biosynthesis and utilization and further enhanced drought performance of females, possibly determining that AA improved drought tolerance more in females than in males. Interestingly, overexpression of acetyl-CoA synthetase (ACS) could reprogram fatty acids, increase LysAc levels, and improve drought tolerance, highlighting the involvement of ACS-derived acetyl-CoA in drought responses. In addition, drought and exogenous AA induced sexually dimorphic LysAc associated with histones, transcription factors, and metabolic enzymes in willows. Especially, exogenous AA may greatly improve the photosynthetic capacity of S. myrtillacea males by decreasing LysAc levels and increasing the abundances of photosynthetic proteins. While hyperacetylation in glycolysis, TCA cycle, and fatty acid biosynthesis potentially possibly serve as negative feedback to acclimate acetyl-CoA biosynthesis and utilization in drought-stressed males and AA-applicated females. Thus, acetyl-CoA biosynthesis and utilization determine the sexually dimorphic responses of S. myrtillacea to drought and exogenous AA.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast.

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.

Nucleus-Translocated ACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy.

Overcoming metabolic stress is a critical step in tumor growth. Acetyl coenzyme A (acetyl-CoA) generated from glucose and acetate uptake is important for histone acetylation and gene expression. However, how acetyl-CoA is produced under nutritional stress is unclear. We demonstrate here that glucose deprivation results in AMP-activated protein kinase (AMPK)-mediated acetyl-CoA synthetase 2 (ACSS2) phosphorylation at S659, which exposed the nuclear localization signal of ACSS2 for importin α5 binding and nuclear translocation. In the nucleus, ACSS2 binds to transcription factor EB and translocates to lysosomal and autophagy gene promoter regions, where ACSS2 incorporates acetate generated from histone acetylation turnover to locally produce acetyl-CoA for histone H3 acetylation in these regions and promote lysosomal biogenesis, autophagy, cell survival, and brain tumorigenesis. In addition, ACSS2 S659 phosphorylation positively correlates with AMPK activity in glioma specimens and grades of glioma malignancy. These results underscore the significance of nuclear ACSS2-mediated histone acetylation in maintaining cell homeostasis and tumor development.

Nuclear Acetyl-CoA Production by ACLY Promotes Homologous Recombination.

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role in promoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice.