Regulatory mechanisms of one-carbon metabolism enzymes.

One-carbon metabolism is a central metabolic pathway critical for the biosynthesis of several amino acids, methyl group donors, and nucleotides. The pathway mostly relies on the transfer of a carbon unit from the amino acid serine, through the cofactor folate (in its several forms), and to the ultimate carbon acceptors that include nucleotides and methyl groups used for methylation of proteins, RNA, and DNA. Nucleotides are required for DNA replication, DNA repair, gene expression, and protein translation, through ribosomal RNA. Therefore, the one-carbon metabolism pathway is essential for cell growth and function in all cells, but is specifically important for rapidly proliferating cells. The regulation of one-carbon metabolism is a critical aspect of the normal and pathological function of the pathway, such as in cancer, where hijacking these regulatory mechanisms feeds an increased need for nucleotides. One-carbon metabolism is regulated at several levels: via gene expression, posttranslational modification, subcellular compartmentalization, allosteric inhibition, and feedback regulation. In this review, we aim to inform the readers of relevant one-carbon metabolism regulation mechanisms and to bring forward the need to further study this aspect of one-carbon metabolism. The review aims to integrate two major aspects of cancer metabolism-signaling downstream of nutrient sensing and one-carbon metabolism, because while each of these is critical for the proliferation of cancerous cells, their integration is critical for comprehensive understating of cellular metabolism in transformed cells and can lead to clinically relevant insights.

Nuclear PTEN Regulates Thymidylate Biosynthesis in Human Prostate Cancer Cell Lines.

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor governs a variety of biological processes, including metabolism, by acting on distinct molecular targets in different subcellular compartments. In the cytosol, inactive PTEN can be recruited to the plasma membrane where it dimerizes and functions as a lipid phosphatase to regulate metabolic processes mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin complex 1 (mTORC1) pathway. However, the metabolic regulation of PTEN in the nucleus remains undefined. Here, using a gain-of-function approach to targeting PTEN to the plasma membrane and nucleus, we show that nuclear PTEN contributes to pyrimidine metabolism, in particular de novo thymidylate (dTMP) biosynthesis. PTEN appears to regulate dTMP biosynthesis through interaction with methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), a key enzyme that generates 5,10-methylenetetrahydrofolate, a cofactor required for thymidylate synthase (TYMS) to catalyze deoxyuridylate (dUMP) into dTMP. Our findings reveal a nuclear function for PTEN in controlling dTMP biosynthesis and may also have implications for targeting nuclear-excluded PTEN prostate cancer cells with antifolate drugs.

Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers.

The most common cancers of the aerodigestive tract (ADT) are non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). The tumor stroma plays an important role in ADT cancer development and progression, and contributes to the metabolic heterogeneity of tumors. Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor stroma of ADT cancers and exert pro-tumorigenic functions. Metabolically, glycolytic CAFs support the energy needs of oxidative (OXPHOS) carcinoma cells. Upregulation of the monocarboxylate transporter 4 (MCT4) and downregulation of isocitrate dehydrogenase 3α (IDH3α) are markers of glycolysis in CAFs, and upregulation of the monocarboxylate transporter 1 (MCT1) and the translocase of the outer mitochondrial membrane 20 (TOMM20) are markers of OXPHOS in carcinoma cells. It is unknown if glycolytic metabolism in CAFs is a driver of ADT cancer aggressiveness. In this study, co-cultures in vitro and co-injections in mice of ADT carcinoma cells with fibroblasts were used as experimental models to study the effects of fibroblasts on metabolic compartmentalization, oxidative stress, carcinoma cell proliferation and apoptosis, and overall tumor growth. Glycolytic metabolism in fibroblasts was modulated using the HIF-1α inhibitor BAY 87-2243, the antioxidant N-acetyl cysteine, and genetic depletion of MCT4. We found that ADT human tumors express markers of metabolic compartmentalization and that co-culture models of ADT cancers recapitulate human metabolic compartmentalization, have high levels of oxidative stress, and promote carcinoma cell proliferation and survival. In these models, BAY 87-2243 rescues IDH3α expression and NAC reduces MCT4 expression in fibroblasts, and these treatments decrease ADT carcinoma cell proliferation and increase cell death. Genetic depletion of fibroblast MCT4 decreases proliferation and survival of ADT carcinoma cells in co-culture. Moreover, co-injection of ADT carcinoma cells with fibroblasts lacking MCT4 reduces tumor growth and decreases the expression of markers of metabolic compartmentalization in tumors. In conclusion, metabolic compartmentalization with high expression of MCT4 in CAFs drives aggressiveness in ADT cancers.

Stem Cell Rejuvenation by Restoration of Youthful Metabolic Compartmentalization.

Stem cell dysfunction is a hallmark of aging. Much recent study suggests that epigenetic changes play a critical role in the loss of stem cell function with age. However, the underlying mechanisms require elucidation. A recent report describes a process by which mild mitochondrial stress associated with aging causes lysosomal-mediated decreases in CiC, the mitochondrial citrate transporter, in bone marrow-derived mesenchymal stem cells (MSCs). This, in turn, results in a deficit of acetyl-CoA in the nucleus and hypoacetylation of histones. The altered epigenome results in skewered stem cell differentiation favoring adipogenesis and disfavoring osteogenesis, which is problematic given the role the MSCs play in maintaining the integrity of bone tissue. Restoration of nuclear acetyl-CoA by either ectopic expression of CiC or acetate supplementation of MSCs in culture rejuvenates the MSC, restoring the potential to efficiently differentiate along the osteogenic lineage. Citrate, which has recently been reported to extend lifespan in Drosophila, chemically incorporates acetyl-CoA and may prove useful to restore cytoplasmic and nuclear acetyl-CoA levels. The general applicability of the CiC defect in old cells, particularly stem cells, should be established.

A large portion of the astrocyte proteome is dedicated to perivascular endfeet, including critical components of the electron transport chain.

The perivascular astrocyte endfoot is a specialized and diffusion-limited subcellular compartment that fully ensheathes the cerebral vasculature. Despite their ubiquitous presence, a detailed understanding of endfoot physiology remains elusive, in part due to a limited understanding of the proteins that distinguish the endfoot from the greater astrocyte body. Here, we developed a technique to isolate astrocyte endfeet from brain tissue, which was used to study the endfoot proteome in comparison to the astrocyte somata. In our approach, brain microvessels, which retain their endfoot processes, were isolated from mouse brain and dissociated, whereupon endfeet were recovered using an antibody-based column astrocyte isolation kit. Our findings expand the known set of proteins enriched at the endfoot from 10 to 516, which comprised more than 1/5th of the entire detected astrocyte proteome. Numerous critical electron transport chain proteins were expressed only at the endfeet, while enzymes involved in glycogen storage were distributed to the somata, indicating subcellular metabolic compartmentalization. The endfoot proteome also included numerous proteins that, while known to have important contributions to blood-brain barrier function, were not previously known to localize to the endfoot. Our findings highlight the importance of the endfoot and suggest new routes of investigation into endfoot function.

Metabolic Compartmentalization at the Leading Edge of Metastatic Cancer Cells.

Despite advances in targeted therapeutics and understanding in molecular mechanisms, metastasis remains a substantial obstacle for cancer treatment. Acquired genetic mutations and transcriptional changes can promote the spread of primary tumor cells to distant tissues. Additionally, recent studies have uncovered that metabolic reprogramming of cancer cells is tightly associated with cancer metastasis. However, whether intracellular metabolism is spatially and temporally regulated for cancer cell migration and invasion is understudied. In this review, we highlight the emergence of a concept, termed "membraneless metabolic compartmentalization," as one of the critical mechanisms that determines the metastatic capacity of cancer cells. In particular, we focus on the compartmentalization of purine nucleotide metabolism (e.g., ATP and GTP) at the leading edge of migrating cancer cells through the uniquely phase-separated microdomains where dynamic exchange of nucleotide metabolic enzymes takes place. We will discuss how future insights may usher in a novel class of therapeutics specifically targeting the metabolic compartmentalization that drives tumor metastasis.

Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma.

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

Intra-Tumoral Metabolic Zonation and Resultant Phenotypic Diversification Are Dictated by Blood Vessel Proximity.

Differential exposure of tumor cells to blood-borne and angiocrine factors results in diverse metabolic microenvironments conducive for non-genetic tumor cell diversification. Here, we harnessed a methodology for retrospective sorting of fully functional, stroma-free cancer cells solely on the basis of their relative distance from blood vessels (BVs) to unveil the whole spectrum of genes, metabolites, and biological traits impacted by BV proximity. In both grafted mouse tumors and natural human glioblastoma (GBM), mTOR activity was confined to few cell layers from the nearest perfused vessel. Cancer cells within this perivascular tier are distinguished by intense anabolic metabolism and defy the Warburg principle through exercising extensive oxidative phosphorylation. Functional traits acquired by perivascular cancer cells, namely, enhanced tumorigenicity, superior migratory or invasive capabilities, and, unexpectedly, exceptional chemo- and radioresistance, are all mTOR dependent. Taken together, the study revealed a previously unappreciated graded metabolic zonation directly impacting the acquisition of multiple aggressive tumor traits.

Lack of evidence for substrate channeling or flux between wildtype and mutant isocitrate dehydrogenase to produce the oncometabolite 2-hydroxyglutarate.

Monoallelic point mutations in the gene encoding the cytosolic, NADP+-dependent enzyme isocitrate dehydrogenase 1 (IDH1) cause increased production of the oncometabolite 2-hydroxyglutarate (2-HG) in multiple cancers. Most IDH1 mutant tumors retain one wildtype (WT) IDH1 allele. Several studies have proposed that retention of this WT allele is protumorigenic by facilitating substrate channeling through a WT-mutant IDH1 heterodimer, with the WT subunit generating a local supply of α-ketoglutarate and NADPH that is then consumed by the mutant subunit to produce 2-HG. Here, we confirmed that coexpression of WT and mutant IDH1 subunits leads to formation of WT-mutant hetero-oligomers and increases 2-HG production. An analysis of a recently reported crystal structure of the WT-R132H IDH1 heterodimer and of in vitro kinetic parameters for 2-HG production, however, indicated that substrate channeling between the subunits is biophysically implausible. We also found that putative carbon-substrate flux between WT and mutant IDH1 subunits is inconsistent with the results of isotope tracing experiments in cancer cells harboring an endogenous monoallelic IDH1 mutation. Finally, using a mathematical model of WT-mutant IDH1 heterodimers, we estimated that the NADPH:NADP+ ratio is higher in the cytosol than in the mitochondria, suggesting that NADPH is unlikely to be limiting for 2-HG production in the cytosol. These findings argue against supply of either substrate being limiting for 2-HG production by a cytosolic IDH1 mutant and suggest that the retention of a WT allele in IDH1 mutant tumors is not due to a requirement for carbon or cofactor flux between WT and mutant IDH1.

Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.

Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both ß-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Compartmentalization and Regulation of Sulfate Assimilation Pathways in Plants.

Plants utilize sulfate to synthesize primary and secondary sulfur-containing metabolites required for growth and survival in the environment. Sulfate is taken up into roots from the soil and distributed to various organs through the functions of membrane-bound sulfate transporters, while it is utilized as the primary substrate for synthesizing sulfur-containing metabolites in the sulfate assimilation pathways. Transporters and enzymes for the assimilative conversion of sulfate are regulated in highly organized manners depending on changes in sulfate supply from the environment and demand for biosynthesis of reduced sulfur compounds in the plant systems. Over the past few decades, the effect of sulfur nutrition on gene expression of sulfate transporters and assimilatory enzymes has been extensively studied with the aim of understanding the full landscape of regulatory networks.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.