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Previews the PRC2 complex as a whole clearly discriminates between specific and nonspecific RNAs and therefore is able to interact selectively with target RNAs (Figure 1). This apparent discrepancy between high binding affinity of EZH2 to RNAs on the one hand and the much more selective binding properties of the PRC2 complex on the other appears to be mediated at least in part by EED. While the subunit alone shows only low affinity to RNA, in complex with EZH2 it reduces the affinity of EZH2 for RNA significantly and might therefore increase the specificity of PRC2 RNA-binding behavior. Interestingly, binding of RNA to PRC2 reduces its methyltransferase activity in an RNA concentration- and binding-affinity-dependent manner: An RNA with high binding affinity to EZH2 (such as RepA) decreases its catalytic activity more than a less-specific binder. Correspondingly, the PRC2 subunit JARID2 can negatively modulate the interaction of core PRC2 to RNA and thereby increases the catalytic activity of the complex. This study nicely shows that the binding affinity and specificity of the PRC2 complex to RNA is not the same as that of its
subcomponents. EZH2 alone shows high binding affinity; its selectivity appears relatively indiscriminate. As part of the PRC2 complex, however, several more layers of regulation are added in the form of at least EED and JARID2. Both help increase the specificity of RNA binding at the cost of binding affinity, which results in reduction of RNA-mediated catalytic inhibition of EZH2. Thus, PRC2bound RNAs not only guide the complex to a target genomic locus, but also play an important role in regulating the methyltransferase activity of EZH2. Although both studies come to somewhat different conclusions, several aspects of their proposed models are similar. For example, in both cases PRC2-dependent and -independent components (e.g., JARID2 or the ‘‘local chromatin context,’’ respectively) are important regulators of PRC2 activity. Also, RNA binding itself is required for recruitment, but also for modulation of EZH2 catalytic activity. Taken together, these studies nicely show that loading of PRC2 to RNA, stable recruitment of the complex to chromatin, and EZH2 catalytic activity can be highly regulated and independent
processes, which are at least in part mediated by modulation of RNA binding affinities of EZH2 and the local chromatin environment. REFERENCES Cifuentes-Rojas, C., Hernandez, A.J., Sarma, K., and Lee, J.T. (2014). Mol. Cell 55, this issue, 171–185. Davidovich, C., Zheng, L., Goodrich, K.J., and Cech, T.R. (2013). Nat. Struct. Mol. Biol. 20, 1250–1257. Di Croce, L., and Helin, K. (2013). Nat. Struct. Mol. Biol. 20, 1147–1155. Khalil, A.M., Guttman, M., Huarte, M., Garber, M., Raj, A., Rivea Morales, D., Thomas, K., Presser, A., Bernstein, B.E., van Oudenaarden, A., et al. (2009). Proc. Natl. Acad. Sci. USA 106, 11667– 11672. Li, G., Margueron, R., Ku, M., Chambon, P., Bernstein, B.E., and Reinberg, D. (2010). Genes Dev. 24, 368–380. Margueron, R., and Reinberg, D. (2011). Nature 469, 343–349. Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, L.H., Helms, J.A., Farnham, P.J., Segal, E., and Chang, H.Y. (2007). Cell 129, 1311–1323. Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J., and Lee, J.T. (2008). Science 322, 750–756.
Localization of NADPH Production: A Wheel within a Wheel Oliver D.K. Maddocks,1 Christiaan F. Labuschagne,1 and Karen H. Vousden1,* 1Cancer Research UK Beatson Institute, Switchback Road, Glasgow G61 1BD, UK *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.molcel.2014.07.001
In this issue of Molecular Cell, Lewis et al. (2014) describe a new method to determine where in the cell NADPH is produced, contributing to a growing appreciation that the THF cycle is an important source of mitochondrial NADPH. The control of metabolism is a fundamental requirement for all life, with perturbations of metabolic homeostasis underpinning numerous disease-associated pathologies. In eukaryotic cells, metabolic reactions can be localized to different organelles, and in several cases the same metabolic pathway is supported
by parallel reactions that are restricted to different cellular compartments. Where a reaction takes place can have a profound impact on the outcome and utilization of the metabolites that are produced and adds a further layer of complexity to our ability to properly understand—and therefore harness—metabolism for the
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improvement of human health. Now a cluster of publications (Fan et al., 2014; Labuschagne et al., 2014; Lewis et al., 2014) has brought the focus onto the compartmentalization and directionality of one-carbon metabolism and how this pathway supports NADPH production for antioxidant defense and reductive
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TTHF HF Serine SSer eriin nee
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Figure 1. Simplified Representation of Mitochondrial and Cytoplasmic THF Cycles Showing NADPH Production at the Step Catalyzed by MTHFD1L and MTHFD1 Using deuterium-labeled serine, electron transfer (denoted by colored stars) can be traced through NADPH and detected in the xenometabolite 2HG. The compartment in which NADPH is produced can be determined depending on whether the mitochondrial (mIDH2) or cytoplasmic (mIDH1) forms of mutant IDH are expressed in the cell. Orange boxes show alternative sources of NADPH in mitochondria and cytoplasm. THF, tetrahydrofolate; me-THF, methenyl/ methylene-THF; aKG, alpha-ketoglutyrate; 2HG, 2-hydroxygutarate; 1C, one-carbon unit; for-THF, formyl-THF.
biosynthetic reactions. Balancing the supply and demand for these two processes is clearly an important feature of cancer cell metabolism. The paper by Lewis et al. (2014) addresses a key question: where in the cell is NADPH made, and how is NADPH production compartmentalized? Generally, the main source of NADPH is thought to be the oxidative pentose phosphate pathway, a cytoplasmic branch of glycolysis that provides both NADPH and ribose for nucleotide synthesis. Since NADPH cannot cross membranes, utilization of this cytosolic NADPH pool for mitochondrial reactions would require multistep shuttling reactions. However, other potential sources of NADPH exist, including the reactions involving different isoforms of malic enzyme and isocitrate dehydrogenase (IDH) (Figure 1). Less appreciated (until now) has been the contribution of serine and glycine metabolism, which, via the tetrahydrofolate (THF) cycle, can be a significant source of NADPH. Serine/glycine metabolism occurs in both the cytosol and the mitochondria, and most of the reactions in these path-
ways can occur in both directions (Figure 1). This is of particular significance when considering NADPH production, since the production of one-carbon units to support thymidylate synthesis would generate NADPH if derived from serine or glycine but consume NADPH if derived from formate (Figure 1). So far, however, the direction of this pathway and the relative contribution of each compartment have been difficult to assess. By developing an elegant new method to trace electrons for NADPH production through the mitochondrial or cytosolic pathways, Lewis et al. (2014) demonstrated that serine/glycine metabolism occurs mainly in the mitochondria, which are the main source of reactive oxygen species (ROS). NADPH production from this pathway could therefore be used to limit mitochondrial ROS, so preventing damage and promoting cell survival. Using 2 H-labeled serine, the authors traced the deuterium onto NADPH and cleverly harnessed mutant isocitrate dehydrogenase (mtIDH), which uses NADPH as cofactor, to trap the electron in 2-hydroxygluterate (2HG). Importantly, 2HG is a xenometabo-
lite in normal cells that is not further metabolized and can therefore serve as a reporter molecule. By generating cell lines that express mutant IDH1 (cytosol) or IDH2 (mitochondria), Lewis et al. (2014) were able to distinguish between cytosolic- or mitochondrial-generated 2HG and from this deduce the compartment of origin of the 2H-serine-derived NADPH. As the authors point out, this reporter system can be used to determine compartment-specific NADPH production in any NADP/NADPH reaction by labeling the electrons of the reaction substrate that will be transferred for NADPH production. In a separate study, Fan et al. (2014) also showed that serine metabolism is a source of NADPH, providing evidence that this can occur in the mitochondria. Furthermore, this study demonstrated a role for mitochondrial glycine metabolism in the generation of mitochondrial NADPH. This is an extremely interesting result, since it suggests that glycine is not used for the production of formate in the mitochondria (through which onecarbon units can be transferred to the
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Previews cytosol for nucleotide synthesis and other reactions) but rather is completely oxidized in the mitochondria to produce additional NADPH. In agreement with these observations, we also found that glycine was not used to generate onecarbon units for nucleotide synthesis (Labuschagne et al., 2014) and, furthermore, that excess glycine had an inhibitory effect on proliferation. By developing a new method to measure metabolic flux—termed ‘‘pulse-stop-flux’’— we found that excess glycine was converted to serine, a reaction that consumes one-carbon units and thus opposes nucleotide synthesis. Interestingly, cells growing in medium containing ample serine showed a net efflux of glycine, suggesting that highly proliferating cells have developed mechanisms to limit the accumulation of intracellular glycine. Only when extracellular serine was depleted did the cells start to consume glycine. A number of interesting questions can now be addressed. Are the reactions leading to the compartmentalized production of NADPH coupled so that NADPH produced from certain pathways is only used in specific reactions? Is the compartmentalization of serine and glycine metabolism to provide NADPH and one-carbon units that has been seen in cancer cell lines also seen in normal tissue? As is common with these studies, most of the work has been carried out in a limited number of cells grown in culture, and the physiological relevance remains to be explored. Previous studies have shown that the key enzymes responsible for NADPH production (or consumption, depending on the direction of the reaction) in both compartments, MTHFD1 and MTHFD2, are essential for normal devel-
opment in mice (Di Pietro et al., 2002; MacFarlane et al., 2009). While the requirement for MTHFD2 supports the importance of the mitochondrial reactions, the role for MTHFD1 in feeding mitochondrially derived formate back into the cytoplasmic one-carbon pool means that these results cannot be used to define a role for cytoplasmic serine metabolism in supporting the THF cycle. Indeed, most of the evidence so far suggests a predominance of the mitochondrial reactions, although there is likely to be some plasticity to allow utilization of the serine metabolism pathways in either compartment, depending on the circumstances. Most obviously, these systems will allow cells to rapidly shift the outcome of the THF cycle to balance their requirements for redox regulation and nucleotide synthesis. The importance of serine and glycine metabolism in cancer development has become very clear over recent years. Many cancer cell lines take up large amounts of exogenous serine (Jain et al., 2012; Maddocks et al., 2013), and the enzymes responsible for de novo serine synthesis are upregulated in several cancer types (Locasale et al., 2011; Possemato et al., 2011). Even though glycine cleavage is not a major contributor of onecarbon units in cell lines (Fan et al., 2014; Jain et al., 2012; Labuschagne et al., 2014), upregulation of the glycine cleavage system enzyme GLDC clearly contributes to tumorigenesis in some tissues (Zhang et al., 2012). The exact role of GLDC in promoting cancer development therefore remains to be determined, and the recent studies provide two interesting and not mutually exclusive explanations; glycine cleavage may be important to detoxify excess intracellular
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glycine and/or to increase mitochondrial antioxidant capacity through NADPH generation. Taken together, these recent publications make great inroads for our understanding of the importance of serine and glycine metabolism in cancer.
REFERENCES Di Pietro, E., Sirois, J., Tremblay, M.L., and MacKenzie, R.E. (2002). Mol. Cell. Biol. 22, 4158– 4166. Fan, J., Ye, J., Kamphorst, J.J., Shlomi, T., Thompson, C.B., and Rabinowitz, J.D. (2014). Nature 510, 298–302. Jain, M., Nilsson, R., Sharma, S., Madhusudhan, N., Kitami, T., Souza, A.L., Kafri, R., Kirschner, M.W., Clish, C.B., and Mootha, V.K. (2012). Science 336, 1040–1044. Labuschagne, C.F., van den Broek, N.J., Mackay, G.M., Vousden, K.H., and Maddocks, O.D. (2014). Cell Rep 7, 1248–1258. Lewis, C.A., Parker, S.J., Fiske, B.P., McCloskey, D., Gui, D.Y., Green, C.R., Vokes, N.I., Feist, A.M., Vander Heiden, M.G., and Metallo, C.M. (2014). Mol. Cell 55, this issue, 253–263. Locasale, J.W., Grassian, A.R., Melman, T., Lyssiotis, C.A., Mattaini, K.R., Bass, A.J., Heffron, G., Metallo, C.M., Muranen, T., Sharfi, H., et al. (2011). Nat. Genet. 43, 869–874. MacFarlane, A.J., Perry, C.A., Girnary, H.H., Gao, D., Allen, R.H., Stabler, S.P., Shane, B., and Stover, P.J. (2009). J. Biol. Chem. 284, 1533–1539. Maddocks, O.D., Berkers, C.R., Mason, S.M., Zheng, L., Blyth, K., Gottlieb, E., and Vousden, K.H. (2013). Nature 493, 542–546. Possemato, R., Marks, K.M., Shaul, Y.D., Pacold, M.E., Kim, D., Birsoy, K., Sethumadhavan, S., Woo, H.K., Jang, H.G., Jha, A.K., et al. (2011). Nature 476, 346–350. Zhang, W.C., Shyh-Chang, N., Yang, H., Rai, A., Umashankar, S., Ma, S., Soh, B.S., Sun, L.L., Tai, B.C., Nga, M.E., et al. (2012). Cell 148, 259–272.
