2019 Zheng

RE VIEW
Toward a better understanding of folate metabolism

in health and disease
Yuxiang Zheng and Lewis C. Cantley
Folate metabolism is crucial for many biochemical processes, including purine and thymidine monophosphate (dTMP)
biosynthesis, mitochondrial protein translation, and methionine regeneration. These biochemical processes in turn support
critical cellular functions such as cell proliferation, mitochondrial respiration, and epigenetic regulation. Not surprisingly,
abnormal folate metabolism has been causally linked with a myriad of diseases. In this review, we provide a historical
perspective, delve into folate chemistry that is often overlooked, and point out various missing links and underdeveloped
areas in folate metabolism for future exploration.
Introduction merged into coherent pathways. Among these enzymes, dihy-

In 1931, Lucy Wills reported that an unusual form of anemia, drofolate reductase (DHFR) was identified as the major target
macrocytic anemia, could be cured by yeast extracts, suggesting of aminopterin and its more widely used close analogue, meth-
that the disease is a nutritional deficiency (Carpenter, 2003a,b). otrexate (Werkheiser, 1961; Huennekens, 1994). In the following
Soon thereafter, many groups were racing to identify the ac- decades, rapid development of molecular biology techniques
tive nutrient, later named folic acid or folate. This was at a time allowed cloning of the genes encoding the enzymes, and Robert
when modern chromatography techniques were yet to be devel- Schimke discovered that amplification of the DHFR gene confers
oped, yet folic acid was successfully isolated by two independent tumor resistance to methotrexate. Remarkably, this was the first
groups using ingenious methods, including using activated char- known case of gene amplification in mammalian somatic cells
coal to adsorb and concentrate folic acid and using a microbio- (Schimke, 1989).
logical assay to rapidly detect its activity (Hutchings et al., 1941; Many excellent reviews on folate metabolism have appeared
Mitchell et al., 1941). These intense efforts culminated in 1948, over the past decade, some of which cover metabolic compart-
when E.L. Robert Stokstad and colleagues at Lederle Laboratories mentation (Tibbetts and Appling, 2010; Scotti et al., 2013), folate
elucidated the chemical structure of folic acid by chemical degra- transport (Zhao et al., 2011), and the roles of folates in redox ho-
dation and total synthesis (Stokstad and Jukes, 1987). meostasis (Ducker and Rabinowitz, 2017), embryonic develop-
Having learned of the discovery of folic acid, Sidney Farber ment (Momb and Appling, 2014), cancers (Locasale, 2013; Yang
recruited a cohort of leukemic children to test whether folic acid and Vousden, 2016), and plants (Hanson and Gregory, 2011).
might be able to restore normalcy to the blood cells in leukemia, It is sometimes claimed in the current literature that all the
just as it does in macrocytic anemia (Farber et al., 1947). Although components in the metabolic pathways have been identified, and
this trial was quickly stopped, as folic acid was found to actually the challenge now is to understand how the metabolic fluxes are
exacerbate the disease, it led to a revised idea that folic acid an- regulated. While we wholeheartedly agree with the latter point,
tagonists might stop leukemia. This revised idea was borne out we share with others the opinion that many components in
in Farber’s next trial. As described in his landmark paper in 1948 mammalian folate metabolism remain to be identified, and their
(Farber and Diamond, 1948), one of the tested folic acid antago- functions remain to be understood. Recently introduced disci-
nists, aminopterin, produced temporary remission in children plines, such as comparative genomics (Gabaldón and Koonin,
with acute lymphoblastic leukemia. Thus, the era of cancer che- 2013), functional genomics (Wang et al., 2014; Shalem et al., 2015;
motherapy had begun. Horlbeck et al., 2016), and organelle proteomics (Chapel et al.,
What also ensued was a “golden era” for enzymology in gen- 2013; Pagliarini and Rutter, 2013; Calvo et al., 2016), have opened
eral (1950–1960; Huennekens, 1996), during which many en- up new opportunities for solving these remaining puzzles. Thus,
zymes involved in folate metabolism were purified from crude in this review, we emphasize missing links and underdeveloped
cell or tissue extracts and characterized, and their reactions areas and discuss potential approaches to illuminate them. We
Department of Medicine, Meyer Cancer Center, Weill Cornell Medicine, New York, NY.
Correspondence to Lewis C. Cantley: lcantley@med.cornell.edu.
© 2018 Zheng and Cantley This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after
the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0
International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
Rockefeller University Press https://doi.org/10.1084/jem.20181965 253

J. Exp. Med. 2019 Vol. 216 No. 2 253–266
begin with a survey of folate chemistry, an important aspect of THF. In the absence of antioxidants, the half-life of THF in
folate metabolism that is sometimes overlooked. aqueous solutions at 37°C is typically <30 min (Pollock and
Kaufman, 1978). The pathway of THF decomposition is mediated
Structure and chemistry of folates by quinoid DHF (qDHF), generated via a free radical mechanism
Nomenclature (Fig. 2 A). qDHF is unstable and readily cleaves into 7,8-dihydrop-
By convention, “folates” is a generic term, referring to a large terin, formaldehyde, and p-aminobenzoylglutamate (pABG; Reed
family of compounds consisting of a 2-amino-4-hydroxy-pteri- and Archer, 1980; Burgos-Barragan et al., 2017). Formaldehyde
dine ring, linked by a methylene (CH2) group to a p-aminoben- is a potent cross-linker for proteins and nucleic acids (Burgos-
zoyl moiety, which is in turn linked through amide bond to the Barragan et al., 2017).
α-amino group of a monoglutamate or poly-γ-glutamate (Fig. 1 A; 10-Formyl-THF. In the absence of antioxidants, 10-formyl-THF
Blakley, 1987). One-carbon (1C) units can be attached to N5, N10, is readily oxidized to 10-formyl-DHF (half-life, typically <30 min
or both. On the other hand, the name “folic acid” is reserved for at 37°C), and on prolonged incubation, is further oxidized to
the synthetic form with the fully oxidized pteridine ring and no 10-formyl-folic acid (Baggott et al., 1995; Baggott, 2000).
1C substitution (Fig. 1 B). 5,10-CH2-THF and 5,10-CH+-THF. There appear to be few reports
The number of distinct molecular species within the folate on the stability of 5,10-CH2-THF and 5,10-CH+-THF, presumably
family could be as many as 150, due to the combinatorial effect because 5,10-CH2-THF is in equilibrium with THF and formal-
of varying 1C and pteridine oxidation states and varying poly- dehyde (Kallen, 1971), and 5,10-CH+-THF would hydrolyze to
glutamate chain lengths (Krumdieck et al., 1983). Nevertheless, 10-formyl-THF and, to a much lesser degree, 5-formyl-THF at
typically <50 species are detectable in natural animal and plant neutral pHs (Stover and Schirch, 1993).
sources (Gregory, 1989), with the pteridine ring predominantly DHF. DHF is much more stable than THF, with a half-life of
in the most reduced, tetrahydro state (Fig. 1 B), N5 and N10 either 4.5 h at 30°C, pH 7.0 (Reed and Archer, 1980). DHF can undergo
unsubstituted or substituted with one of six different 1C units either pteridine ring oxidation to folic acid or C9-N10 cleavage
(Fig. 1 C), and the polyglutamate chain length being predomi- to pABG and 6-formyl-dihydropterin or dihydroxanthopterin
nantly five to eight (Lin et al., 1993). (Chippel and Scrimgeour, 1970; Reed and Archer, 1980). The
partition into these three pathways seems to depend on the
Folates as 1C carriers or electron carriers pH and the type of buffer (Chippel and Scrimgeour, 1970; Reed
In most folate-mediated reactions, tetrahydrofolate (THF) serves and Archer, 1980).
as a 1C carrier, which obtains the 1C unit from a donor (e.g., ser- 5-Methyl-THF. 5-Methyl-THF can be readily oxidized to
ine) and then transfers it to an acceptor, typically a biosynthetic 5-methyl-5,6-DHF. In the presence of thiols or ascorbate, 5-meth-
intermediate. The attached 1C units exist in three different oxi- yl-5,6-DHF readily reverts to 5-methyl-THF. Conversely, when
dation states that are equivalent to methanol, formaldehyde, and subject to more oxidative conditions, 5-methyl-5,6-DHF either
formate (Fig. 1 C) and can be interconverted by cellular enzymes rearranges to a 5-methyl-dihydro-pyrazino-s-trazine compound
using nicotinamide adenine dinucleotide phosphate (NADP) or or cleaves into an unidentified pterin and pABG (Gregory, 1989).
nicotinamide adenine dinucleotide (NAD) as a cosubstrate. Folic acid and 5-formyl-THF. These two species are stable to ox-
Although the pteridine ring in folates is capable of undergoing idation, except under harsh conditions such as in the presence of
redox reactions, this capability is rarely used. A notable exception KMnO4 (Maruyama et al., 1978).
occurs in the thymidylate synthase–catalyzed reaction (Fig. 1 B),
in which the folate pteridine ring undergoes a two-electron oxi- Paradoxical stability of folates in vivo
dation. To sustain THF levels, therefore, DHFR is required to act Given the intrinsic tendency of reduced folates to decompose,
downstream of thymidylate synthase to recycle DHF back to THF. it is surprising that folates were found to be extremely slowly
turned over in human whole body or in cultured mammalian
Chemical lability of reduced folates cells (Krumdieck et al., 1978; von der Porten et al., 1992; Lawrence
Naturally occurring, reduced folates are chemically labile. In fact, et al., 2014). For example, using a 2H-folic acid tracer, folate
the first identified folate, folic acid, was likely an artifact due to breakdown rates in adult humans were estimated to be ∼0.5%
the lengthy isolation procedures, during which natural folates per day (half-life, ∼19 d; von der Porten et al., 1992). This is in
were largely decomposed by reacting with molecular oxygen, and stark contrast to the situation in plants, where folate breakdown
a minor component among the oxidized products was folic acid is very rapid, at a rate of ∼10% per day (Orsomando et al., 2006).
(Krumdieck et al., 1983). The chemical lability is an important as- A series of excellent reviews (Linster et al., 2013; Lerma-Ortiz
pect of folates, not only presenting a technical challenge for their et al., 2016; Sun et al., 2017) emphasized two important facets of
chemical analysis, but also reflecting their tendency to break metabolism that were largely overlooked in classic textbooks of
down in vivo. Antioxidants, ascorbate in particular, are effective biochemistry. First, enzymes are not always exquisitely specific
in minimizing folate decomposition and have proved useful in and efficient, and many of them also catalyze unwanted side re-
various analytical methods for folates (Quinlivan et al., 2006). actions. Moreover, many metabolites are chemically labile and
Different classes of folates, defined by the pteridine oxidation reactive, and thus prone to damage. To counter such metabolite
state and the 1C identity, display markedly different stabilities damage, cells have often evolved dedicated metabolite repair en-
and follow distinct degradation pathways. We describe the path- zymes. In an example related to folate metabolism, 5-formyl-THF,
ways for each class, roughly in the order of increased stability. a dead-end metabolite formed from 5,10-CH+-THF by a side reac-
Zheng and Cantley Journal of Experimental Medicine 254

Folate metabolism in health and disease https://doi.org/10.1084/jem.20181965
Figure 1. Folate chemistry. (A) Chemical struc-
ture of THF. (B) Redox reactions for the pteridine
ring of folates. (C) The 1C unit is derived from
donors such as serine, exists in three different
oxidation states, and is used for various bio-
chemical processes.

Figure 2. Folate decomposition and damage repair. (A) Chemical pathway of THF decomposition. (B) Cellular enzymatic systems for folate damage repair.
tion of serine hydroxymethyltransferases (SHMT1/2; Stover and Folate pathways: Identified and unidentified components
Schirch, 1992) or spontaneously at mildly acidic pHs (Baggott, Among the three processes we discuss in this section, folate up-
2000), is salvaged by methenyl-THF synthetase (MTHFS) back take and catabolism typically occur on a much slower timescale
to 5,10-CH+-THF in eukaryotes and most prokaryotes (Fig. 2 B; than most 1C transfer reactions, consistent with the cell’s differ-
Linster et al., 2013). ential needs for folate and 1C. Folate is needed only in catalytic
We recently found that cellular THF decomposition can be amounts to function as a cofactor, whereas 1C must be rapidly
prevented by an enzyme, quinoid dihydropteridine reductase transferred via heavy-traffic pathways to support demanding
(QDPR); specifically, the qDHF intermediate of THF decomposi- processes such as nucleotide synthesis.
tion can be reduced by QDPR back to THF at the expense of two
reducing equivalents from NADH (Fig. 2 B; Zheng et al., 2018). Folate uptake and intracellular accumulation
Moreover, the oxidation product of 10-formyl-THF, 10-formyl- Folate uptake at the cell surface. At the plasma membrane, two
DHF, can be reduced back to 10-formyl-THF by DHFR, or to transmembrane carriers (SLC19A1 and SLC46A1) and three gly-
THF by tandem actions of ATIC and DHFR (Fig. 2 B; Zheng et cosylphosphatidylinositol-anchored receptors (FOLR1–3) can
al., 2018). Thus, the oxidative damage to the two least stable fo- mediate folate uptake (Fig. 3 A; Zhao et al., 2011). The substrate
lates, THF and 10-formyl-THF, can be enzymatically repaired in of these transporters and receptors is folate monoglutamate,
mammalian cells. consistent with the fact that the circulating folate is predomi-
It is likely that other factors also contribute to folate sta- nantly 5-methyl-THF monoglutamate (Ratanasthien et al., 1974).
bility in vivo. For example, folates can be stabilized through SLC19A1, also known as the reduced folate carrier, is a widely ex-
protein binding (Wittwer and Wagner, 1981; Cook and Wagner, pressed antiporter that exchanges a reduced folate for another
1982; Min et al., 1988; Jones and Nixon, 2002). In addition, anion substrate. Candidates for the counter substrate of SLC19A1,
the reduced intracellular milieu maintained by glutathione- including thiamine phosphates and 5-aminoimidazole-4-carbox-
and thioredoxin-mediated systems may play an indispens- amide ribonucleotide monophosphate (ZMP or AICAR), have
able role as well. been suggested (Visentin et al., 2012). ZMP is an intermediate

in purine biosynthesis and is known to accumulate in folate de- accumulation. In cells lacking FPGS, folate monoglutamates that
ficiency, in both bacteria (Stetten and Fox, 1945; Bochner and have been taken up will readily efflux out of the cell, resulting in
Ames, 1982) and mammal cells (Herbert et al., 1964; McGeer et al., only ∼10% of normal folate accumulation (Osborne et al., 1993).
1965). Therefore, using ZMP as a counter substrate for SLC19A1 Thus, polyglutamation adds multiple negative charges to the
would represent an elegant feedback mechanism for regulating imported folates and thereby dramatically enhances their in-
intracellular folate homeostasis. tracellular accumulation. Moreover, polyglutamation markedly
SLC46A1, also known as the proton-coupled folate transporter, increases the affinity of most folate-dependent enzymes for their
is most active at acidic extracellular pHs. Consistent with its low folate substrate (Matthews et al., 1987).
pH optimum, SLC46A1 catalyzes symport of protons with the fo- DHF, THF, and 10-formyl-THF are efficient substrates for
late substrate, notably in the acidic, upper region of the small in- FPGS, whereas 5-methyl-THF, 5-formyl-THF, and folic acid are
testine, where it is highly expressed. Loss-of-function mutations poor substrates (Cichowicz and Shane, 1987). Because 5-meth-
in SLC46A1 are the only known cause of the rare disease hered- yl-THF is the major circulating folate, its conversion by methi-
itary folate malabsorption (Qiu et al., 2006; Zhao et al., 2007). onine synthase (MTR) to THF is thought to be the first metabolic
The folate receptors, strongly expressed in certain epithelial step in cells that precedes polyglutamation (Cook et al., 1987).
tissues and some gynecological cancers, mediate folate uptake FPGS is expressed in most tissues as at least two isoforms that
through an endocytic pathway, but the exact mechanism is a mat- result from alternative splicing and alternative translational start
ter of debate (Paulos et al., 2004; Chadda et al., 2007). Whatever sites, one of which targets to the mitochondrial matrix (Lawrence
the mechanism, to support cellular folate-dependent functions, et al., 2014). This enables the establishment of distinct, nonmis-
the internalized folate monoglutamate must be effluxed from an cible cytosolic and mitochondrial folate pools that operate in two
internal vesicular compartment into the cytosol (Fig. 3 A). It has parallel pathways, described next.
been suggested that SLC46A1 could mediate this efflux (Zhao
et al., 2009), yet the same authors also pointed out that FOLR1 Parallel cytosolic and mitochondrial 1C pathways
can function normally in some cells that lack SLC46A1 expres- Parallel 1C pathways exist in the cytosol and in mitochondria, re-
sion, indicating that there is at least one alternative transporter spectively, each composed of a core set of serine hydroxymethyl-
(Zhao et al., 2011). transferase, CH2-THF dehydrogenase/CH+-THF cyclohydrolase,
Folate uptake into the mitochondria. At the mitochondrial and 10-formyl-THF synthetase activities (Fig. 3 B; Shin et al.,
inner membrane, SLC25A32, a member of the mitochondrial 2014). These two pathways are connected by small metabolites
carrier family that consists of ∼50 proteins, imports reduced that can readily traverse the mitochondrial inner membrane,
folate monoglutamates into the mitochondrial matrix (Fig. 3 A). such as serine, glycine, and formate. The consensus for the in-
Surprisingly, however, human SLC25A32 cDNA was able to com- terrelationship of the two pathways is that the mitochondrial 1C
plement the growth defects of a yeast mutant lacking a homol- pathway produces formate, which in turn is used in the cytosolic
ogous gene, FLX1, that encodes a putative mitochondrial flavin pathway for purine, dTMP, and methionine biosynthesis, or ex-
adenine dinucleotide (FAD) transporter (Spaan et al., 2005). creted out of the cell if in excess (Christensen and MacKenzie,
Recently, compound heterozygous mutations in SLC25A32 were 2006; Tibbetts and Appling, 2010; Meiser et al., 2016; Ducker and
found in a patient with riboflavin-responsive exercise intoler- Rabinowitz, 2017).
ance, further supporting a link between SLC25A32 and FAD Recently, SFXN1 has been identified as a mitochondrial ser-
metabolism (Schiff et al., 2016). Thus, these studies have raised ine transporter (Kory et al., 2018). SLC25A38 has been suggested
the question of whether mammalian SLC25A32 is a carrier for to be a mitochondrial glycine transporter (Fernández-Murray
folate, FAD, or both. et al., 2016; Lunetti et al., 2016). However, the genes encoding
The virtual absence of mitochondrial folates in SLC25A32 mitochondrial transporters for formate, sarcosine, and dimeth-
mutant Chinese hamster ovary (CHO) cells strongly argues that ylglycine remain unknown (Fig. 3 A).
folate is a bona fide substrate for SLC25A32 (Titus and Moran, Another well-recognized role for the mitochondrial 1C path-
2000; McCarthy et al., 2004). In contrast, FLX1-deficient yeast way is glycine synthesis (Titus and Moran, 2000; Christensen
still has sizable mitochondrial FAD (Tzagoloff et al., 1996). More- and MacKenzie, 2006; Tibbetts and Appling, 2010; Jain et al.,
over, mammalian mitochondria (Barile et al., 2000; Calvo et al., 2012; Ducker and Rabinowitz, 2017). A long-standing question is
2016), and possibly yeast mitochondria as well (Bafunno et al., why the cytosolic SHMT1 cannot produce sufficient glycine. To
2004), can synthesize FAD from riboflavin using riboflavin ki- generate glycine continuously via SHMT1, THF has to be contin-
nase and FAD synthetase, so it is not apparent why FAD needs to uously regenerated. For the cytosolic 1C pathway operating in the
be imported from the cytosol. Given that the transport mecha- direction of glycine synthesis, THF regeneration is impeded by
nism of SLC25A32 remains to be further delineated (Lawrence et the unfavorable NADP/NADPH and ADP/ATP ratios in the cytosol
al., 2011), it is possible that SLC25A32 is an antiporter that cou- (Veech et al., 1979; Christensen and MacKenzie, 2006). Our recent
ples folate import with FAD export (Fig. 3 A). Consistent with this work showed that overexpression of ALDH1L1 can overcome this
possibility, yeast FLX1 has been suggested to be a mitochondrial thermodynamic barrier by bypassing the unfavorable 10-formyl-
FAD export carrier (Bafunno et al., 2004). THF synthetase step, thereby allowing continuous THF regen-
Cytosolic and mitochondrial folate retention by polyglutamation. eration and glycine synthesis in the cytosol (Zheng et al., 2018).
Folate polyglutamation, catalyzed by folylpolyglutamate synthe- The mitochondrial 1C pathway also supports mitochon-
tase (FPGS), plays an indispensable role for intracellular folate drion-specific functions. It is well known that mitochondrial

Figure 3. Folate transport and metabolic compartmentation. (A) Knowledge gaps in cellular folate metabolism. (B) Parallel cytosolic and mitochondrial
folate pathways.

10-formyl-THF is used for N-formylation of the initiator methi- its folate-binding activity was confirmed by biophysical experi-
onine in mitochondrial methionyl-tRNAMet. Loss-of-function ments (Teplyakov et al., 2004). YgfZ homologues (named IBA57
mutations in the human formyltransferase gene, MTFMT, have for the human gene) occur in all domains of life and, through
been causally linked to Leigh syndrome, a mitochondrial dis- a poorly understood mechanism, are essential for the assem-
ease (Tucker et al., 2011). Unexpectedly, a recent study showed bly or maintenance of certain iron-sulfur proteins in bacteria
that SHMT2-knockout HCT116 cells contained normal levels (Waller et al., 2010, 2012) and in yeast (Gelling et al., 2008) and
of formylmethionine in mitochondrially translated peptides human (Sheftel et al., 2012) mitochondria (Fig. 3 A). At least in
(Morscher et al., 2018). In contrast, another recent study found bacteria, the function of YgfZ in iron-sulfur cluster metabolism
severe depletion of mitochondrial formyl-methionyl-tRNAMet in is folate dependent (Waller et al., 2010, 2012). Further inves-
SHMT2-knockout Jurkat cells (Minton et al., 2018). It remains to tigation into IBA57 is much needed to fully decipher the mito-
be clarified whether these discrepancies can be accounted for by chondrial respiration defects caused by impaired mitochondrial
the differences in the cell line context, and if so, what the con- folate metabolism.
text-specific modulators are.
Recent research is also beginning to reveal a surprising role Folate catabolism
for mitochondrial 5,10-CH2-THF in mitochondrial protein trans- Folate catabolism is extremely slow under normal conditions, but
lation. These studies, first performed on prokaryotic systems, can be greatly accelerated with methotrexate treatment (Zheng
highlight the synergy between genetic, structural, and biochem- et al., 2018). The bulk of folate catabolism in vivo is accounted for
ical approaches. It was first established, through genetics, that by the appearance of p-acetamidobenzoylglutamate (pAcABG)
two bacterial proteins, MnmE and MnmG, are involved in modi- and pterins, products of folate C9-N10 bond cleavage, in urine
fying uridine at the wobble position of certain bacterial transfer (Murphy et al., 1976; Krumdieck et al., 1978). Whereas the C9-
RNAs (tRNAs) to 5-methyluridine derivatives (Elseviers et al., N10 bond cleavage is probably spontaneous, reflecting folate in-
1984; Hagervall et al., 1987), yet the biochemical understanding trinsic instability, formation of pAcABG from the direct cleavage
was lagging behind. product pABG requires an enzyme, the NAT1 acetyltransferase
A breakthrough came when x-ray crystallography revealed (Minchin, 1995; Wakefield et al., 2007).
that MnmE contains a folate-binding fold strikingly resem- The cellular pathways for folate catabolism are poorly un-
bling that in several known folate-binding enzymes (Scrima et derstood. Because plasma membrane efflux is prohibited for
al., 2005; Scrutton and Leys, 2005). This revelation enabled the polyglutamated folates, pABG, or pAcABG, conversion of folate
long-awaited in vitro reconstitution experiments, in which vari- polyglutamate into folate monoglutamate, or deglutamation,
ous THF derivatives were tested (together with other known cos- is required for cellular clearance of folate catabolites. The only
ubstrates) for the ability to stimulate the reaction of the MnmE/ known enzyme capable of folate deglutamation is γ-glutamyl-hy-
MnmG complex with tRNAs (Moukadiri et al., 2009; Armengod drolase (GGH), which is either contained within lysosomes or se-
et al., 2012). Indeed, 5,10-CH2-THF was found to be active and was creted into the extracellular space (Hugonnet, 2012). Consistent
suggested to be an essential cofactor for MnmE/MnmG. with the role of GGH in folate deglutamation, GGH up-regulation
The human orthologues of MnmE and MnmG, GTPBP3 and was associated with a shift in intracellular folate distribution to
MTO1, have been predicted to be responsible for the occurrence shorter polyglutamate species in a hepatoma cell line (Yao et al.,
of 5-taurinomethyluridine at the wobble position in certain mi- 1995). To access GGH, polyglutamated folates, pABG, or pAcABG
tochondrial tRNAs (Suzuki et al., 2011; Suzuki and Suzuki, 2014). have to be imported into the lysosome. A lysosomal carrier spe-
To date, successful in vitro reconstitution for this putative activ- cific for polyglutamated rather than monoglutamated metho-
ity has not been published. However, compelling genetic evidence trexate was reported in the 1990s (Barrueco et al., 1992; Sirotnak
has been provided by a recent study: knocking out either MTO1 or and Tolner, 1999), but many questions remain unanswered, such
SHMT2 (which supplies mitochondrial 5,10-CH2-THF) in HCT116 as its gene identity and whether it preferentially imports cleaved
cells resulted in no detectable levels of 5-taurinomethyluridine folates (Fig. 3 A). The acidic pH (∼4.5) of lysosomal lumen is ex-
in mitochondrial tRNAs (Morscher et al., 2018). Consistent with pected to precipitate some spontaneous reactions of folates, such
the crucial role of this wobble uridine modification in stabiliz- as the conversion of 5,10-CH+-THF to 5-formyl-THF (Baggott,
ing codon–anticodon pairing, these knockout cells were defec- 2000). Finally, it remains to be determined whether the mono-
tive in mitochondrial protein translation (Morscher et al., 2018). glutamate product of GGH is directly secreted via lysosomal exo-
Thus, the mitochondrial folate pathway provides the 5,10-CH2- cytosis (Settembre et al., 2013) or first released into the cytosol
THF substrate for GTPBP3/MTO1-mediated mitochondrial tRNA via a lysosomal carrier.
modification in human cells, an important finding with rele-
vance to the mitochondrial disorders myoclonic epilepsy with Approaches to identify missing components
ragged red fibers and mitochondrial encephalomyopathy, lactic Historically, many components involved in folate metabolism
acidosis, and stroke-like episodes, which are caused by defective were discovered by biochemical purification to chase down the
5-taurinomethyluridine modification (Suzuki et al., 2011; Suzuki protein responsible for an activity of interest (Tan et al., 1977;
and Suzuki, 2014). Huennekens, 1996) and by forward genetic screens followed by
Our understanding of mitochondrial folate metabolism is not cDNA complementation (Dixon et al., 1994; Titus and Moran,
yet complete, however. Like MnmE, another bacterial protein 2000). The use of radioactive folic acid tracer in rats also greatly
YgfZ contains a folate-binding fold in its crystal structure, and accelerated the discoveries of folate transporters/receptors and

folate-binding enzymes for which folate was not an expected
substrate but later proved to be an integral component of their
catalysis (Wagner, 1982; Garcia et al., 2016). More recently,
searching for paralogs in the then just-completed human genome
database allowed identification of missing genes for known ac-
tivities (Prasannan et al., 2003; Bolusani et al., 2011).
It is possible that saturation has not been reached in tradi-
tional forward genetic screens where chemical-induced mu-
tagenesis could have target bias. Moreover, in some cases, the
causal gene remains elusive. For example, glycine auxotrophic
mutants of CHO cells discovered in the 1970s (Chasin et al., 1974;
McBurney and Whitmore, 1974) fall into four complementation
groups: GLYA (=SHMT2), GLYB (=SLC25A32), AUXB1 (=FPGS),
and GLYC/AUXB2. Although the growth defect of the GLYC mu-
tant was complemented by human DHFR2 (also named DHFRL1)
cDNA (Anderson et al., 2011), the DHFR2 gene does not exist in
the rodent from which the CHO line was derived. Thus, the GLYC/
AUXB2 gene remains to be identified, for example by RNA or
whole-exome sequencing.
The radioactive folic acid tracer approach had been limited to
folate binders that are highly abundant and show high affinity
(dissociation constant = submicromolar or lower) toward its fo- Figure 4. The folate cycle is linked to the methionine cycle via MTHFR
late ligand (Min et al., 1988). The same limitations would be en- and vitamin B12–using MTR.
countered if a folate affinity column (Wittwer and Wagner, 1980)
were used to pull down potential binders (Burdine and Kodadek,
2004). Although protein abundance is less an issue with the est, one may zero in on short stretches of protein-encoding or
advent of highly sensitive mass spectrometry instruments, the promoter sequences and correlate the protein or DNA sequences
requirement for tight binding remains a hurdle. Thus, new pro- with well-defined protein kinase phosphorylation or transcrip-
teomic approaches such as thermal proteome profiling (Martinez tion factor binding motifs, as detailed in previous reviews (Frame
Molina et al., 2013; Franken et al., 2015) and limited proteolysis– and Cohen, 2001; Manning and Cantley, 2007) and exemplified by
small molecule mapping (Piazza et al., 2018), developed to cap- some recent studies (Ben-Sahra et al., 2013, 2016; Ye et al., 2014;
ture both strong and weak protein–ligand interactions in cells, DeNicola et al., 2015; Lee et al., 2017). Ultimately, the hypotheses
can be powerfully complementary. thus generated need to be tested using genetic, biochemical, and
If an activity is known to localize to an intracellular organelle pharmacological approaches.
(e.g., lysosomes or mitochondria), then the search for the gene
can be dramatically narrowed down to dozens or fewer candi- Interaction of the folate cycle with the methionine cycle
dates by referring to published inventories of genes encoding ATP, NAD, and NADP are universal cofactors in metabolism.
proteins for that organelle (Chapel et al., 2013; Pagliarini and Therefore, folate pathways are connected to almost every other
Rutter, 2013; Calvo et al., 2016). A caveat is that these invento- metabolic pathway through shared ATP, NAD, and NADP pools.
ries have incomplete coverage and finite false discovery rates Although folate pathways can contribute to the production of
(Pagliarini and Rutter, 2013), and moreover, for the lysosome, NADPH and ATP, their fluxes are also directly affected by com-
it might be difficult to distinguish resident proteins from cargo partment-specific ATP/ADP, NAD/NADH, and NADPH/NADP
proteins delivered there for degradation (Chapel et al., 2013). ratios. Recently it has been proposed that NADPH produced by
Nevertheless, recent success stories, such as the discoveries of ALDH1L2 in the mitochondrial 1C pathway is important to limit
the mitochondrial pyruvate carrier (Bricker et al., 2012; Herzig mitochondrial ROS (Piskounova et al., 2015; Yang and Vousden,
et al., 2012) and the mitochondrial calcium uniporter (Baughman 2016). Here, we further discuss the methionine cycle, which
et al., 2011), highlight the excellent potential for this approach. is linked to folate cycle via a more “specialized” metabolite,
The above organelle proteomics–based approach correlates an 5-methyl-THF (Fig. 4).
activity with a list of proteins on the basis of subcellular colocal- The link between folate and methionine cycles has long been
ization. Yet there are other ways to infer correlations. For exam- recognized, since a deficiency in either folate or vitamin B12
ple, genes with unknown functions can be linked to genes with (an essential cofactor for MTR) produces megaloblastic ane-
well-defined functions on the basis of coevolution, coexpression, mias that are hematologically indistinguishable (Shane and
protein–protein interactions, suppression and synthetic interac- Stokstad, 1985). The megaloblastic anemia in both cases is as-
tions, and for microbial orthologues of a candidate human gene, cribed to defective DNA synthesis and can be reversed by large
gene clustering and gene fusion (https://string-db.org/ and doses of folates. Thus, vitamin B12 deficiency causes functional
https://thebiogrid.org/; Pagliarini and Rutter, 2013; Niehaus et folate deficiency, and this is best explained by the methyl trap
al., 2015). To further understand the function of a gene of inter- model (Shane and Stokstad, 1985). Methylenetetrahydrofolate

reductase (MTHFR)–catalyzed conversion of 5,10-CH2-THF to (Shane and Stokstad, 1985). In some other cell types, folate defi-
5-methyl-THF is irreversible in cells, due to the large negative ciency can cause uracil misincorporation into DNA, leading to
standard free-energy change and high cytosolic NADPH/NADP chromosome breakage (Blount et al., 1997). It has been proposed
ratios (Matthews and Drummond, 1990). In addition, methionine that such breaks could contribute to the increased risk of cancer
synthase is the only enzyme that can remove the methyl group (Blount et al., 1997).
from 5-methyl-THF. Thus, a lack of vitamin B12 and thus MTR Cerebral folate deficiency, caused by folate receptor autoan-
activity would irreversibly trap folates into 5-methyl-THF, at the tibodies or germline mutations in FOLR1 or SLC46A1, is mani-
expense of 5,10-CH2-THF and 10-formyl-THF, both of which are fested in neurological impairments and shows some resemblance
crucial to DNA synthesis. to severe MTHFR deficiency (Watkins and Rosenblatt, 2014).
The original methyl trap model has been expanded to include Thus, folate metabolism plays a crucial, yet poorly defined,
two subsequent developments. First, MTHFR is allosterically role in the brain.
inhibited by S-adenosyl-methionine (SAM) with an inhibition Insufficient folate intake around and during pregnancy has
constant of 2.8 µM (Shane and Stokstad, 1985). Thus, large doses been associated with increased risk of neural tube defects, which
of methionine can increase intracellular SAM, block the methyl is the basis for the mandatory folate fortification of foods in the
trap, and thereby ameliorate both folate and B12-deficient mega- US since 1998 (Momb and Appling, 2014). Strong evidence for the
loblastic anemia. Second, as discussed above, the MTR-catalyzed role of the mitochondrial folate pathway in neural tube develop-
conversion of 5-methyl-THF to THF is likely the first folate met- ment has been provided by a MTHFD1L-knockout mouse model,
abolic step in cells that precedes polyglutamation, so vitamin B12 which presents neural tube defects with 100% penetrance while
deficiency would also compromise intracellular folate polyglu- on a folate-proficient diet (Momb et al., 2013). In addition, mice
tamation and retention. This explains the clinically observed, deficient in the AMT or GLDC gene, encoding a subunit of the
diminished total cellular folate content in vitamin B12 deficiency. glycine cleavage system, also developed neural tube defects with
It is currently unresolved to what extent the folate cycle con- partial penetrance (Narisawa et al., 2012; Pai et al., 2015). It was
tributes to cellular methylations in normal physiology and dis- further shown with the MTHFD1L or GLDC-knockout mice that
eases such as cancers. MTHFR-knockout mice are viable, and supplementation with formate rescued the neural tube defects
some clinical symptoms of human MTHFR deficiency may be (Momb et al., 2013; Pai et al., 2015). In a recent study, further
ascribed to hyperhomocysteinemia rather than alterations in deletion of MTHFR in GLDC-null mouse embryos resulted in
methylation (Schwahn et al., 2003). Many cultured cell lines are significant protection from neural tube defects, suggesting that
unable to proliferate when the medium methionine is replaced folate and formate-supported nucleotide synthesis, rather than
with homocysteine (Borrego et al., 2016; Lien et al., 2017), and MTHFR-mediated methyl group biogenesis, is crucial for neural
indeed stable isotope labeling and mass spectrometry analysis tube development (Leung et al., 2017).
indicated that for many cell lines, the contribution of serine and Conversely, excess folate intake may enhance proliferation
folate pathways to the active methyl group is insignificant com- of malignant tumors (Strickland et al., 2013). Moreover, genes
pared with medium methionine (Pike et al., 2010; Shyh-Chang encoding enzymes in serine biosynthesis and the mitochondrial
et al., 2013; Ducker et al., 2016; Maddocks et al., 2016). Under- folate pathway are often amplified or overexpressed in can-
standing the mechanistic bases for such methionine dependency cers (Locasale et al., 2011; Possemato et al., 2011; Nilsson et al.,
in cancer cell lines should be an important future direction, es- 2014). It has been suggested that metabolic activities under on-
pecially in light of the recent discoveries that many oncogenic cogenic control in cancers can be categorized based on whether
mutations directly impact DNA and histone methyltransferases they are transforming, enabling, or neutral (Vander Heiden and
and demethylases (Garraway and Lander, 2013). DeBerardinis, 2017). As attested by the successes of methotrexate
and other antifolates in treating cancers, it is likely that serine
Functions of folate metabolism and disease connections biosynthesis and mitochondrial folate metabolism belong to the
The biochemical outputs of folate metabolism include nucleotide first two categories, and therefore are potential therapeutic tar-
synthesis, serine-glycine interconversion, mitochondrial tRNA gets (Mullarky et al., 2016; Pacold et al., 2016; Ducker et al., 2017).
modification, and methyl group biogenesis, which in turn sup- On the other hand, restriction of dietary serine and glycine has
ports cellular functions such as proliferation, mitochondrial res- been proposed to treat cancers that lack up-regulation of de novo
piration, and epigenetic regulation. Abnormal folate metabolism serine biosynthesis (Maddocks et al., 2017).
has been causally linked with a myriad of diseases. However, for
each disease, it is often unclear which biochemical processes and Future perspectives
cellular functions are culprits. Throughout this review, we have highlighted areas that await
The earliest known folate-related disease is probably meg- further development and discussed how to explore them using
aloblastic anemia. Systemic folate deficiency, whether dietary, existing technologies. Yet it is hard to predict where upcoming
alcohol or drug induced, or due to germline mutations in DHFR technologies will lead us. From a biochemical perspective, we en-
or SLC46A1 (Qiu et al., 2006; Banka et al., 2011; Cario et al., 2011), vision a future where the entire human proteome is structurally
invariably results in megaloblastic anemia. The molecular basis solved (Petsko, 2014) and the folate binding fold (Scrutton and
for megaloblastic anemia has been ascribed to deranged DNA Leys, 2005) might be revealed in many more proteins, thus push-
synthesis, which results in chromosome breakage, and contin- ing forward a new level of understanding of folate metabolism in
ued cell growth without division during red blood cell production human health and disease.

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This work was supported by a Department of Defense Blount, B.C., M.M. Mack, C.M. Wehr, J.T. MacGregor, R.A. Hiatt, G. Wang, S.N.
Breast Cancer Research Program postdoctoral fellowship Wickramasinghe, R.B. Everson, and B.N. Ames. 1997. Folate deficiency
(W81XWH-13-1-0251 to Y. Zheng), a National Cancer Institute causes uracil misincorporation into human DNA and chromosome
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grant (388207 to L.C. Cantley), and a gift from the Mindy and Jon Bochner, B.R., and B.N. Ames. 1982. ZTP (5-amino 4-imidazole carboxamide
Gray family (L.C. Cantley). riboside 5′-triphosphate): a proposed alarmone for 10-formyl-tetra-
L.C. Cantley is a founder and member of the Science Advisory hydrofolate deficiency. Cell. 29:929–937. https://doi.org/10.1016/0092
-8674(82)90455-X
Board of Agios Pharmaceuticals and Petra Pharmaceuticals, com- Bolusani, S., B.A. Young, N.A. Cole, A.S. Tibbetts, J. Momb, J.D. Bryant, A.
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ceuticals provides funds to support the laboratory of L.C. Cantley. mitochondrial methylenetetrahydrofolate dehydrogenase isozyme ex-
pressed in adult tissues. J. Biol. Chem. 286:5166–5174. https://doi.org/10
Y. Zheng declares no competing financial interests. .1074/jbc.M110.196840
Author Contributions: Y. Zheng and L.C. Cantley conceived Borrego, S.L., J. Fahrmann, R. Datta, C. Stringari, D. Grapov, M. Zeller, Y. Chen,
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RE VIEW

Toward a better understanding of folate metabolism

Introduction merged into coherent pathways. Among these enzymes, dihy-

Correspondence to Lewis C. Cantley: lcantley@med.cornell.edu.

Rockefeller University Press https://doi.org/10.1084/jem.20181965 253

Zheng and Cantley Journal of Experimental Medicine 254

Zheng and Cantley Journal of Experimental Medicine 255

Zheng and Cantley Journal of Experimental Medicine 256

Zheng and Cantley Journal of Experimental Medicine 257

Zheng and Cantley Journal of Experimental Medicine 258

Zheng and Cantley Journal of Experimental Medicine 259

Zheng and Cantley Journal of Experimental Medicine 260

Zheng and Cantley Journal of Experimental Medicine 261

Zheng and Cantley Journal of Experimental Medicine 262

Zheng and Cantley Journal of Experimental Medicine 263

Zheng and Cantley Journal of Experimental Medicine 264

Zheng and Cantley Journal of Experimental Medicine 265

Zheng and Cantley Journal of Experimental Medicine 266

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2019 Zheng

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2019 Zheng

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2019 Zheng

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RE VIEW

Toward a better understanding of folate metabolism

Introduction merged into coherent pathways. Among these enzymes, dihy-

Correspondence to Lewis C. Cantley: lcantley@​med​.cornell​.edu.

Rockefeller University Press https://doi.org/10.1084/jem.20181965 253

Zheng and Cantley Journal of Experimental Medicine 254

Zheng and Cantley Journal of Experimental Medicine 255

Zheng and Cantley Journal of Experimental Medicine 256

Zheng and Cantley Journal of Experimental Medicine 257

Zheng and Cantley Journal of Experimental Medicine 258

Zheng and Cantley Journal of Experimental Medicine 259

Zheng and Cantley Journal of Experimental Medicine 260

Zheng and Cantley Journal of Experimental Medicine 261

Zheng and Cantley Journal of Experimental Medicine 262

Zheng and Cantley Journal of Experimental Medicine 263

Zheng and Cantley Journal of Experimental Medicine 264

Zheng and Cantley Journal of Experimental Medicine 265

Zheng and Cantley Journal of Experimental Medicine 266

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Correspondence to Lewis C. Cantley: lcantley@med.cornell.edu.