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few arrows missing - AIR CARBOXYLASE AND CAIR MUTASE
Source of Atoms in IMP Rings
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IMP to ATP and GTP
Now Pyrimidine Synthesis
Now thymidine synthesis (a dexoynucleotide)
“INPUTS” OF ONE-CARBON METABOLISM
As mentioned above, one-carbon metabolism acts as an integrator of the cell nutrient status by redistributing carbon groups from certain aminoacids, usually serine and glycine, (called “inputs”) to generate various compounds (“outputs”) that serve as building blocks for cell biosynthesis and also maintain the redox and methylation states of cells . Serine can be obtained exogenously (i.e. imported from outside of the cell) as well as endogenously by de novo synthesis (see details below and in Figure Figure1).1). Glycine can be also transported through the plasma membrane . Alternatively, it can be generated from serine through an enzymatic conversion in either cytoplasm or mitochondria. Furthermore, glycine can also be synthesized from threonine as was shown for mouse embryonic stem cells .
Schematic representation of the compartmentalization and enzymatic reactions of one-carbon metabolism
One-carbon metabolism acts as a gauge of the cell nutrient status by redistributing carbon groups from serine and glycine, called “inputs”, to generate various compounds, called “outputs” (shown in black boxes) that serve as building blocks for cell biosynthesis. Also, they maintain the redox and methylation states of cells. Serine and Glycine can be imported through the membrane (shown as green layer) into the cells or it can be synthesized from the intermediate of glycolysis – 3-PG. Metabolic cycles are denoted as circles. Critical enzymes are shown in red. Carriers of one-carbon groups are shown in yellow. For example, 5,10-methyleneTHF provides one-carbons for thymidylate synthesis, catalyzed by the enzyme called Tymidylate Synthase. The positions of one-carbons used for the synthesis of purines (C2, C4, C5, and C8 carbons of purine rings) are indicated. Folate cycle is tightly connected with Methionine cycle. Folate cycle operates both in the cytoplasm and in mitochondria (magenta colored circle) and are linked through Tetra Hydro Folate (THF).
In theory, both serine and glycine can be potential donors of 1C-groups for one-carbon metabolism. However, the actual relationship between serine and glycine metabolism is far more complex. The integrated scheme summarizing the crosstalk of serine and glycine metabolic pathways is presented in Figure Figure11.
There are evidences that cancer cells usually demonstrate increased serine and glycine biosynthesis and uptake [13, 16, 20, 21]. De novo serine synthesis consists of three steps and involves the conversion of 3-phosphoglycerate (3-PG, an intermediate of glycolysis) to 3-phosphopyruvate (3-PP) by the Phosphoglycerate Dehydrogenase (PHGDH) (Figure (Figure1).1). The next step involves conversion of 3-PP to 3-phosphoserine (3-PS) which is mediated by the Phosphoserine Aminotransferase (PSAT1) using glutamate for this transamination. As the final step, the phosphate ester is hydrolyzed by the Phosphoserine Phosphatase (PSPH), resulting in production of serine. Apparently, different cancer cells promote expression of the corresponding enzymes to increase the biosynthesis of serine [13, 21, 22].
It has been shown that cancer cells utilize up to 10% of glycolytic intermediate 3-PG for serine biosynthesis . PHGDH is amplified in a number of cancers, including 6% of breast cancers and 40% of melanomas . Moreover, experiments using siRNA demonstrated that attenuation of PHGDH expression was associated with slow cell growth of non-malignant cells. On the contrary, ectopic expression of PHGDH in the non-cancerous MCF10A breast epithelial cell line disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation .
Besides serine, which represents a critically important “input” of one-carbon metabolism and nucleotide biosynthesis, there is another important metabolite generated at the transamination step of serine biosynthesis - α-ketoglutarate (αKG) . αKG is the entry point through which glutamine supplies carbon to the tricarboxylic acid (TCA) cycle during cell growth, enabling the production of a number of essential biosynthetic precursors .
Taken these results together, it is evident that serine is a hub of one-carbon metabolism and therefore its overexpression is an important feature of different malignancies.
Whereas the importance of serine for the enhanced proliferation of cancer cells is generally accepted, the impact of glycine on this process is the topic for intense debates.
Jain with colleagues  applied a mass-spectrometry approach to measure the consumption and release of 219 metabolites from the media across the NCI-60 panel of cancer cell lines and combined these data with the pre-existing atlas of gene expression. The integrated analysis identified glycine consumption as well as expression of the mitochondrial glycine biosynthetic pathway (SHMT2, MTHFD2 and MTHFD1L) to be strongly correlated with the rates of proliferation across all cancer cell lines.
However, other works have shown that cancer cells fail to consume glycine when serine is abundant [20, 27]. For instance, Labuschange (2014)  showed that cancer cells preferentially consumed serine rather than glycine, and the high level of serine uptake paralleled with glycine efflux. Moreover, according to their results, the excess of glycine even inhibited cell growth. Biochemically, high levels of glycine inhibited the metabolic transformation of the former into purines, required for DNA replication, by driving instead the intracellular glycine-to-serine conversion. Glycine was converted to serine at the expense of 5,10-methylenTHF, thus depleting its intracellular pool and hence slowing the cell growth. Based on these data, it has been proposed that cancer cells release the excess of glycine thereby limiting its intracellular concentration to facilitate serine uptake and serine-to-glycine conversion . Notably, it is the serine-to-glycine conversion process that yields the 5,10-methyleneTHF metabolite required for the maintenance of maximal levels of nucleotide synthesis and proliferation. Serine is converted to glycine by two isoforms of Serine hydroxymethyltransferase (SHMT1 and SHMT2) that correspond to the cytosolic and mitochondrial forms, respectively. The excess amount of glycine, the product of this reaction, can reverse this reaction and therefore should be removed from the cell . On the related note is the fact that two different cancer in vivo models also demonstrated that excess of dietary glycine inhibited the development of tumors [29, 30]. Collectively, glycine biosynthesis is deemed as the central process which sustains one-carbon metabolism and rapid proliferation.
As mentioned above, glycine metabolism is intimately linked with purine biosynthesis and defines the sensitivity to mycophenolate, tiazofurin, alanosine and other inhibitors of purine biosynthesis . In general, glycine can contribute to purine biosynthesis in two ways: by direct incorporation into the purine ring or indirectly, by providing one-carbon units for biochemical reactions involved in the purine ring biosynthesis. The latter can be derived by either synthesis of glycine from serine (the SHMT-catalyzed reaction), or alternatively, by glycine degradation (oxidization to CO2 by a highly evolutionary conserved glycine cleavage system - GCS) (Figure (Figure1).1). It is important to note that the incorporation of glycine into purine nucleotides does not involve oxidation by GCS but is rather mediated by SHMT-derived glycine . These data suggest that SHMT-catalyzed glycine synthesis together with direct incorporation of glycine into the purine rings link glycine production with purine biosynthesis.
As mentioned above, the glycine degradation is exerted by GCS. This system consists of four mitochondrial proteins: the T-protein (GCST or AMT (aminomethyltransferase)), P-protein (GLDC (glycine dehydrogenase)), L-protein (GCSL or DLD (dihydrolipoyl dehydrogenase)), and H-protein (GCSH). It converts glycine to CO2 in the following reaction: Glycine + THF + NAD+ = 5,10-methylene-THF + CO2 + NH3 + NADH2 . Importantly, the products of this enzymatic reaction, 5,10-methyleneTHF and NADH, are required for the nucleotide biosynthesis.
Apparently, GCS is critical for efficient elimination of the excess of glycine. Components of GCS, especially GLDC, are frequently overexpressed in different malignancies and this is linked with cancer progression. Tedeschi and colleagues  showed that about 28% of lung-, 19% of breast-, 9% of prostate-, 30% of colorectal-, 23% of brain- and 21% of ovarian cancers exhibit a significant up-regulation of the 1C-metabolism gene signature, including GLDC. The data from other groups support the notion on overexpression of GCS components in cancer [12, 16].
Zhang and colleagues  have established GLDC as instrumental for the growth and tumorigenesis of tumor-initiating cells derived from the primary NSCLC. Overexpression of GLDC promotes cellular transformation and induces dramatic changes in glycolysis and serine/glycine metabolism, leading to changes in pyrimidine metabolism and cancer cell proliferation. Furthermore, its aberrant regulation often correlates with poor survival of lung cancer patients.
Another study  has shown that GCS may cooperate with SHMT2 to ensure survival and progression of tumors. For example, GLDC-mediated cleavage of the excessive amount of glycine supported the growth of glioma cells with active SHMT2 under ischemic conditions. On the contrary, when the activity of GLDC was inhibited, cells with high levels of SHMT2 were selectively killed. This is explained by the fact that the excess of glycine generated by SHMT2 was subsequently converted into two toxic molecules, aminoacetone and methylglyoxal, which normally are metabolized by GLDC .
Taken these data together, one can reckon that despite the fact that glycine biosynthesis is the hub of one-carbon metabolism, the excess of glycine itself can be detrimental to the tumor cell proliferation and needs to be strictly controlled by either its export or by GCS-mediated clearance.
FOLATE AND METHIONINE CYCLES
The “core” part of the one-carbon metabolism comprises the Folate and Methionine cycles, which are linked together. These two cycles integrate cell nutrient status using 1C-groups from glycine and serine as “inputs” to generate different “outputs” such as nucleotides, glutathione, SAM, and other metabolites, which are required for DNA and RNA biosynthesis, as well as for the maintenance of the redox and epigenetic cell states.
Folates are referred to the family of B9 vitamins . They are naturally present in different sources of food or can be synthesized chemically (e.g. folic acid) as dietary supplements. Folates function as carriers that distribute one-carbon groups from “inputs” to “outputs” (Figure (Figure11 and and2).2). Once transported to the cell, the vitamin undergoes covalent modification by polyglutamination. It is further substituted by the one-carbon moiety in the N5 and/or N10 position at different oxidation levels: formate (10-formylTHF), formaldehyde (5,10-methyleneTHF), or methanol (5-methylTHF) .
Folate cycle, its “outputs” and the energy balance
Critical enzymes of the Folate cycle are shown. TetraHydroFolate (THF) is a carrier that distributes one-carbon groups (1C-group) from serine to different “outputs” – thymidylates, purines, SAM, GSH, etc (shown in black boxes). After accepting the 1C-group, THF undergoes modifications that alter its oxidation states: 10-formylTHF, 5,10-methyleneTHF, 5-methylTHF (shown in different background colors). Donated carbon and nitrogen atoms corresponding to their numbers in the pyrimidine and purine rings are shown in brackets. Red asterisks indicate the enzymes that are currently being explored as drug targets. Enzymes marked with orange asterisks are considered as potential drug targets. Folate cycle can provide cells with additional source of energy. Two molecules of NADPH are synthesized in cytoplasm in reactions catalyzed by DHFR (conversion of DHF to THF) and MTHFD1 (conversion of 5,10-methylenTHF to 5,10-methenylTHF), as well as in mitochondria by MTHFD2L (conversion of 5,10-methylenTHF to 5,10-methenyl THF). One molecules of NADPH is used by MTHFR which links Folate cycle to the Methionine cycle. Also, ATP can be synthesized during MTHFD1- (cytoplasm) or MTHFD1L-mediated (mitochondria) conversion of 10-formylTHF to THF.
As mentioned above, there are only two direct sources of 1C-groups in one-carbon metabolism – serine and glycine. Thus, the central reaction of the Folate cycle is conversion of serine to glycine by SHMT1 and SHMT2 enzymes. By transferring the 1C-group from serine and THF, this reaction generates 5,10-methyleneTHF – the first donor of one-carbon group in the folate cycle. Another source of 5,10-methyleneTHF comes from the enzymatic cleavage of glycine by an enzyme called glycine decarboxylase (GLDC), which resides in mitochondria.
In turn, 5,10-methyleneTHF can be used in three ways (Figure (Figure2).2). First, it can serve as 1C-donor for the initial step of thymidylate biosynthesis, a reaction catalyzed by thymidylate synthase (TS). In this reaction 5,10-methyleneTHF provides one-carbon group for the pyrimidine biosynthesis and is oxidized into dihydrofolate (DHF). In the next reaction dihydrofolate reductase (DHFR) reduces DHF to THF enclosing this metabolic loop.
Second, 5,10-methyleneTHF can be used by a cytosolic enzyme Methylenetetrahydrofolate reductase 1 (MTHFD1), or mitochondrial tandem enzymes Methylenetetrahydrofolate reductases MTHFD2L/MTHFD2, to generate 10-formylTHF. 10-formylTHF is a 1C-donor for the two reactions of purine biosynthesis catalyzed by Trifunctional enzyme Phosphoribosylglycinamide Formyltransferase/ Synthetase/ Phosphoribosylaminoimidazole Synthetase (GART) and Bifunctional 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC), both of which in turn generate THF.
Third, 5,10-methyleneTHF is used by Methylentetrahydrofolatereductase (MTHFR) to generate methylTHF. The latter donates a methyl group to homocycteine resulting in the formation of methionine and THF. By this way the Folate cycle is coupled with Methionine cycle. Finally, THF is converted into 5,10-methyleneTHF by SHMT1 and SHMT2 thus enclosing the Folate cycle.
Another arm of the 1C-metabolic process is the methionine cycle (Figure (Figure3).3). It starts with methionine synthesis from homocysteine and methylTHF catalyzed by methionine synthase (MS). Subsequently, methionine adenyltransferase (MAT) synthesizes SAM, the main donor of methyl groups in the cell. After demethylation, SAM is converted to S-adenosylhomocysteine (SAH). Finally, S-adenosyl homocysteine hydrolase (SAHH) mediates de-adenylation of SAHH resulting in homocysteine and full turn of the cycle.
Folate cycle is coupled with Methionine cycle
During Folate cycle MTHFR reduces 5,10-methyleneTHF to 5-methylTHF. Subsequently, 5-methylTHF donates its carbon group to convert homo-cysteine (hcystein) to methionine by methionine synthase (MS), hence initiating Methionine cycle. In turn, methionine is used by methionine adenosyltransferase (MAT) to generate S-adenosylmethionine (SAM) – the principal donor of methyl groups for DNA and proteins methylation. Thus, SAM is used by different methyltransferases, resulting in S-adenosylhomocysteine after its demethylation. Finally, S-adenosylhomocysteine hydrolase (SAHH) mediates deadenylation of S-adenosylhomocysteine to hcysteine, enclosing the methionine cycle. Homocysteine can be used by cystathionine synthase (CBS), which converts it to cystathionine. In turn, cystathionine is a substrate for cystathionine gamma-lyase (CTH), which uses it for synthesis of cysteine. Cysteine is required for the synthesis of proteins as well as for generation of taurine and glutathione, the latter is one of the critical molecules for redox homeostasis.
“OUTPUTS” OF ONE-CARBON METABOLISM
The Folate and Methionine cycles mediate redistribution of 1C-groups results in biosynthesis of a number of important compounds including nucleotides, several aminoacids, and GSH and SAM molecules that are critical for the maintenance of cell redox status and epigenetic homeostasis (Figure (Figure1).1). All of these compounds are necessary for the rapid proliferation of cancer cells.
The biosynthesis of nucleotides
One of the main outputs of one-carbon metabolism is biosynthesis of nucleotides. This is one of the molecular processes that constrains quickly proliferating cancer cells rate-limiting as it provides building blocks for DNA synthesis, purines and pyrimidines. The biosynthesis of both purine and pyrimidine (thymidylate) nucleotides requires cofactors generated through 1C-metabolism pathways.
The pyrimidine ring is composed of three fragments: C4 to C6 and N1 atoms are provided by aspartate, whereas C2 is derived from HCO3−, while N3 – from glutamine. The central precursor for generating pyrimidines is uridine-monophosphate (UMP). At first, the pyrimidine ring is constructed followed by conjugation with phospho-ribosyl-pyrophosphate (PRPP) (for details see Figure Figure4A).4A). The synthesis of UMP does not require the 1C-cofactors. UMP through UDP can be than converted to either dUMP (for subsequent dTTP synthesis) or other nucleotides UTP, CTP and dCTP. The conversion of dUMP to dTMP is mediated by thymidylate synthase (TS) and requires 5,10-methylenTHF as 1C-donor. This reaction is very important for nucleotide biosynthesis and TS is a target for several approved drugs in cancer therapy (see below).
Major pathways of the nucleotide biosynthesis
A. Shown are the reactions of the biosynthesis of pyrimidines: The participating enzymes are shown in red. The input compounds, intermediates and the resulting products are indicated (future positions of atoms in the nucleotide are indicated in brackets). Reactions are numbered in sequential order. Thus, reactions 1, 2 and 3 are catalyzed by tri-functional enzyme Carbamoyl Dehydrogenase (CAD); 4 – Dihydroorotate Dehydrogenase (DHODH);5 and 6 – bifunctional Uridine Monophosphate Synthetase (UMPS); 7,8,9 - Nucleoside Diphosphate Kinase (NME); 10 - Thymidylate synthase (TS), 11 - CTP Synthase (CTPS). Red asterisks indicate the enzymes that are currently being explored as drug targets. Enzymes marked with orange asterisks are considered as potential drug targets. B. The biosynthesis of purines. Abbreviations are the same as in part A.
The purine ring is composed of different components (see Figure Figure4B):4B): glycine, which is the most used precursor (donates C4, C5 and N7 atoms), HCO3− - the donor of C6, glutamine (the donor of N3 and N9 atoms), aspartate (the donor for N1), and 1C-cofactor - N10-formylTHF which is a donor for C2 and C8. The central intermediate for purines is inosine-monophosphate (IMP). In contrast to pyrimidines, the purine synthesis starts with PRPP and subsequent step-by-step construction of the purine ring (for details see Figure Figure4).4). The IMP generation from PRPP needs five enzymes, three of which are multifunctional (GART, PAICS, ATIC). Reactions, catalyzed by GART and ATIC (the adding atoms C2 and C8 for purine ring) require N10-formylTHF produced by MTHFD1 and MTHFD2 during the folate cycle.
IMP can be converted in the two-steps reactions either into AMP or GMP. The gateway to guanine nucleotides is controlled by IMPDH, making it an “enzyme of consequence” for virtually every organism. This reaction is the first rate-limiting step in guanine nucleotide biosynthesis (Figure (Figure4B4B).
Thus, the biosynthesis of nucleotides is critical for rapidly proliferating cancer cells to ensure the timely DNA replication and therefore represents a promising target for anticancer therapy (see below).
One-carbon metabolism and nucleotide biosynthesis stand in one line with such cancer-related metabolic alterations as increased glycolysis, pentose-phosphate pathway and an acquired ability of de novo synthesis of fatty acids. According to the growing body of evidences, these metabolic features are common to different types of tumors and are considered now as one of the “hallmarks of cancer” [4, 6, 169]. They provide metabolic plasticity to cancer cells which has an impact on different features such as gene expression , epigenetic control  and drug resistance . One carbon metabolism provides “building blocks” (nucleotides, certain aminoacids) as well as contributes to epigenetic (SAM for DNA and protein methylation) and redox (glutathione) homeostasis for rapidly proliferating cancer cells.
The high importance of one-carbon metabolism for cancer cells is reflected in more than 60 years application of its inhibitor (MTX) for a cancer treatment. But, one can notice, that despite the a relatively big number of enzyme operating in one-carbon metabolism and nucleotide biosynthesis, only a few of them are currently used as anticancer targets. They are only: DHFR, TS and RNR. Recently, IMPDH inhibitors became to be used and it will be interesting to see in next years if they are efficient to treat tumors.
Thus, an important question is whether other enzymes of one-carbon metabolism and nucleotide biosynthesis can be efficiently targeted by anticancer therapies without excessive toxicity to normal cells? For instance, as discussed above, there are number of potent GART inhibitors, but none of them has successfully passed through clinical trials due to their high toxicity. A related question is whether it is possible in general to target GART without cytotoxicity to normal cells?