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ScienceDirect Metabolic pathway compartmentalization: an underappreciated opportunity? Annalisa Zecchin1,2, Peter C Stapor1,2, Jermaine Goveia1,2 and Peter Carmeliet1,2 For eukaryotic cells to function properly, they divide their intracellular space in subcellular compartments, each harboring specific metabolic activities. In recent years, it has become increasingly clear that compartmentalization of metabolic pathways is a prerequisite for certain cellular functions. This has for instance been documented for cellular migration, which relies on subcellular localization of glycolysis or mitochondrial respiration in a cell type-dependent manner. Although exciting, this field is still in its infancy, partly due to the limited availability of methods to study the directionality of metabolic pathways and to visualize metabolic processes in distinct cellular compartments. Nonetheless, advances in this field may offer opportunities for innovative strategies to target deregulated compartmentalized metabolism in disease. Addresses 1 Laboratory of Angiogenesis & Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium 2 Laboratory of Angiogenesis & Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium
metabolic pathways are compartmentalized in time and space to support specific cellular processes. The realization that metabolism controls cell function in health and disease has lead to a resurgent interest in cellular metabolism, with the ultimate goal of targeting metabolism therapeutically. However, it is clear that a better understanding of how metabolic compartmentalization contributes to cell function could help to identify novel, perhaps even more specific, therapeutic approaches. We will not provide an all-encompassing literature survey, but rather discuss representative examples that highlight the importance of metabolic compartmentalization to support cell function.
How and why is metabolism compartmentalized?
Introduction
Physical delimitation of metabolic pathways within membrane-delineated organelles or the cytosol is a classical example of metabolic compartmentalization [3]. Metabolic pathways can be localized in distinct domains, even within one compartment. For instance, in smooth muscle cells, glycogen stores are metabolized via oxidative phosphorylation, while external glucose is used for glycolysis [4]. Metabolic enzymes can be also sequestered to scaffold proteins, as demonstrated by the association of glycolytic enzymes with the actin cytoskeleton [5,6,7–9]. Another feature of metabolic enzymes is their ability to assemble in large quaternary structures, ranging from homodimers (fatty acid synthase) and polymers (glutamine synthetase) to multiprotein metabolon complexes (purinosome) [10–13]. Spatial organization of enzyme complexes facilitates transfer of metabolites from one enzyme to the next, thus generating an efficient assembly line and increasing overall metabolic output [5,14,15]. In addition to spatial compartmentalization, metabolic pathways are also temporally compartmentalized. A prime example is the transient peak in glycolysis and glutaminolysis during the S phase of the cell cycle [16].
Given the complexity of biological systems in multicellular organisms, evolution has organized living matter into structured compartments. At the macroscopic level, the human body is divided in distinct organs and tissues. Organs are composed of multiple cell types, cells are divided into organelles, and organelles in turn are defined by spatial architecture. At the molecular level, cellular metabolism is a prime example of an organized complex system. Scientific reductionism has lead to increasingly better descriptions of cellular metabolic networks [1]. However, cells are not simple bags of enzymes and metabolites with random spatial and temporal organization [2]. On the contrary,
There are many benefits to subcellular compartmentalization. First, subcellular compartmentalized microenvironments favor optimal activity of specific enzymes. For instance, peroxisomal oxidase and catalase activity are higher at alkaline pH [17], as is the case in peroxisomes [18,19]. Second, some metabolic reactions produce toxic intermediates that can be contained and disposed of in closed compartments. The containment of hydrogen peroxide and its breakdown by catalase inside peroxisomes is a prime example [20]. Third, compartmentalization can limit diffusion and prevent escape of intermediates
Corresponding author: Carmeliet, Peter ([email protected])
Current Opinion in Biotechnology 2015, 34:73–81 This review comes from a themed issue on Systems biology Edited by Sarah Maria Fendt and Costas D Maranas
http://dx.doi.org/10.1016/j.copbio.2014.11.022 0958-1669/# 2014 Elsevier Ltd. All rights reserved.
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through organelle membranes as is the case for ATP and ADP in muscle cells, which therefore need adenine nucleotide translocase to cross the inner mitochondrial membrane [21]. Fourth, compartmentalization counteracts futile cycles of metabolites that might otherwise be continuously interconverted in reactions operating in opposite directions. In yeast, differential expression of metabolic enzymes periodically occurs over time. Such a precise orchestration regulates energy production through activation of distinct metabolic pathways in restricted temporal windows [22]. Finally, metabolic compartmentalization often increases the efficiency of chemical reactions, as many reactions in free solution would be otherwise diffusion-limited due to restricted local enzyme substrate availability. In brain and muscle, glycogen and glycogenolytic enzymes are compartmentalized together in microdomains to facilitate glycogen breakdown [23]. In the smooth muscle cell example, the internal glycogen stores that are metabolized via oxidative phosphorylation supply energy for actin–myosin contractions, while the external glucose supplies energy for ion transport via glycolysis [24]. Despite the many benefits and obvious necessity of metabolic compartmentalization, its functional role in supporting biological processes is underappreciated. In the remainder of this review, we will provide examples of how metabolic compartmentalization controls specific cell functions.
Meeting energy demand by metabolic pathway compartmentation An example illustrating how metabolic pathways influence biological processes is the hydra, a primitive animal in which distinct metabolic pathways are selectively preferred depending on the type of locomotion adopted. The slow movement of its body over the sea floor relies on oxidative metabolism, while glycolysis is required for the sudden and dynamic changing movements of its tentacles for praying [25]. Likewise, an association of glycolytic enzymes with structures for rapid motility has been described in the tail of sperm cells [26]. Because glycolysis has the capacity for quick ATP production relative to oxidative phosphorylation, it is used for activities requiring high, local levels of ATP over a short period, whereas oxidative phosphorylation more efficiently produces ATP over time. Compartmentalization of glycolysis during cell motility
Cellular migration relies on dynamic remodeling of the cytoskeleton, a process requiring large amounts of local ATP [27,28]. Interaction of glycolytic enzymes with the actin cytoskeleton has been documented in mammalian neurons and skeletal muscle cells as well as in fruitflies and fish muscles. Compartmentalization of glycolysis on the actin cytoskeleton enables efficient and high local ATP production [5,7–9]. The importance of this association is illustrated by the fact that, despite expression of the full complement of active glycolytic enzymes in flight muscles, transgenic fruitflies are unable to fly when Current Opinion in Biotechnology 2015, 34:73–81
glycolytic enzymes fail to colocalize in actin-rich sarcomeres [9]. Migrating endothelial cells rely on highly dynamic actin architectures at the leading edge, lamellipodia and filopodia, which pull the cell body forward [29]. In quiescent endothelial cells, mitochondria and glycolytic enzymes are present in the perinuclear cytosol [6]. In contrast, when these cells start to migrate, they relocate their glycolytic machinery to the lamellipodia and filopodia (Figure 1) [6]. Notably, glycolytic enzymes physically interact and coconcentrate with actin fibers at the membrane ruffles in the lamellipodia, and even become superactivated by this actin association [6]. Interestingly, endothelial cell lamellipodia and filopodia are too narrow and too thin to accommodate bulky mitochondria [6]. Local glycolysis allows rapid generation of ATP for dynamic motility, and prevents taxing ATP demands throughout the cell [6]. Suppressing glycolysis reduces endothelial cell motility, and lamellipodia and filopodia formation, whereas blocking mitochondrial respiration does not affect endothelial cell migration [6]. Similar to ECs, cancer cells often have a high glycolytic flux, which has been implicated in cancer cell invasion. Invasive cancer cells migrate through extracellular matrix using protrusive structures called invadopodia [30]. Proteomic analysis of invadopodia revealed that they are enriched in glycolytic enzymes [31]. High glycolytic flux has accordingly been associated with mesenchymal cancer cell motility and cytoskeleton remodeling, possibly accounting for a more aggressive phenotype [84]. Furthermore, reducing glycolysis in cancer cells decreases their ability to degrade the matrix [31,32]. Mitochondrial compartmentalization during cell motility
Mitochondria are also important for migration of various cancer cells, though this requirement is dependent on the cancer cell type. For instance, invadopodia in human glioma contain active mitochondria [33]. In migrating epithelial cancer cells, mitochondria reside at the leading front, and altering their anterior position impairs migration [34]. In metastatic breast cancer cells, mitochondrial fragmentation promotes, while mitochondrial fusion reduces lamellipodia formation and cell migration [35]. Consistently, inhibition of mitochondrial ATP production reduces migration and actin polymerization, implicating mitochondria as the source of ATP for actin remodeling [35]. Lymphocytes exhibit a profoundly different mode of motility compared to endothelial and cancer cells. In order to migrate in a directional manner, they undergo polarization and generate specialized cell domains. They form a lamellipodium at the leading edge, containing the actin polymerization machinery and chemoattractant receptors, and a uropod at the trailing edge, concentrating adhesion receptors and the microtubule-organizing center (MTOC) www.sciencedirect.com
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Figure 1
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Metabolic compartmentalization during cell motility. (a) Glycolytic ATP production resides throughout the perinuclear cytosol in quiescent, nonmotile ECs (left). In angiogenic ECs (right), glycolytic enzymes compartmentalize in lamellipodia and filopodia at the leading front to generate high levels of ATP for actin polymerization. (b) In unpolarized, nonmotile lymphocytes, mitochondria are distributed at the contact zone of adhesion on substratum (left). During directional movement, mitochondria relocalize to the trailing uropod to supply ATP for actomyosin contraction.
that facilitate migration [36]. This change in cell shape enables them to convert cytoskeletal forces into net cell body displacement. At the uropod, myosin filaments contract actin filaments to provide the tension required for migration [36]. In nonmotile lymphocytes, mitochondria are present at the plasma membrane, close to the contact site between activator and effector cells [37]. When lymphocytes are migrating, mitochondria are transported to the uropod alongside microtubules in a process requiring mitochondrial fission (Figure 1). This strategic position allows mitochondria to supply sufficient local amounts of ATP for the high energy-demanding actomyosin contractions, thereby enabling uropod retraction and cell displacement [38]. The role of glycolysis compartmentalization in lymphocyte migration has not been studied yet. Thus, www.sciencedirect.com
endothelial, cancer and immune cells all differ in their metabolic requirements and compartmentalization during migration. Understanding the differences in compartmentalized metabolism across various cell types (e.g. endothelial versus cancer cells) may provide advantages for designing therapies to target selected cell types. Compartmentalized ATP generation in sustaining neural function
Cells have not only capitalized on the different glycolytic and mitochondrial ATP production dynamics in supporting cytoskeletal dynamics but also in many other processes. For example, compartmentalization of metabolism in neurons is required for synaptic transmission and intracellular transport. Synaptic transmission consumes large Current Opinion in Biotechnology 2015, 34:73–81
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amounts of ATP for synchronized vesicle release, vesicle recycling and generation of synapse potentials [39]. Accordingly, localization of mitochondria to synaptic terminals provides resident hubs for the synthesis of ATP required for synaptic transmission (Figure 2) [40]. Production of ATP by mitochondria is also critical for axon branching of sensory neurons. In neurons, the machinery for protein translation that is necessary for actin-dependent branching is located at axon branching points, but mRNA translation that promotes branching occurs only at axonal locations where mitochondria stall and produce ATP (Figure 2) [41].
demonstrate that neurons compartmentalize different metabolic pathways for specific vesicle related functions, it will be interesting to determine whether this is also true in other cell types and whether other vesicular processes, like endocytosis, are supported by compartmentalized metabolism.
Location-dependent functions of metabolic enzymes Besides being essential for energy production, macromolecular biosynthesis and redox homeostasis in the cytosol or mitochondria, metabolic enzymes can have signaling roles in the nucleus. For instance, emerging evidence supports a link between metabolism and gene transcription. Indeed, levels of acetyl-CoA, the substrate of histone acetyl-transferases, regulate the expression of genes through histone acetylation [47,48]. While cytosolic ATP-citrate lyase (ACLY) is critical for the conversion of citrate to acetyl-CoA, necessary for lipogenesis [49], nuclear localization of ACLY suggests a role in the production of acetyl-CoA in the nucleus, where it can be used for histone acetylation (Figure 3) [48]. Linking nutrient availability to production of acetyl-CoA, ACLY has been correlated to the transcription of genes involved in adipocyte differentiation [48], proliferation in cancer
In contrast, glycolysis provides fuel for fast axonal transport of vesicles to nerve terminals (Figure 2) [42]. Vesicles, which traffic along microtubules and carry trophic factors, possess glycolytic machinery to generate their own ATP to fuel dynein and kinesin transport motors [42]. In addition, glycolysis is compartmentalized at the plasma membrane to fuel high energydemanding ion pumps in neurons [43,44], and in filopodia of astrocytes, glycogen stores have been identified in filopodia-like cellular protrusions [45], likely to release lactate in the extracellular space for oxidative metabolism by neighboring neurons [46]. Although these studies Figure 2
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Metabolic compartmentalization in neural functions. Glycolytic enzymes associate with transported vesicles to provide ATP for motor proteins (a). Local mitochondrial ATP production supports synaptic vesicle release (b) and mRNA translation at axonal branch points (c). Current Opinion in Biotechnology 2015, 34:73–81
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Location-dependent functions of metabolic enzymes. Metabolic enzymes can perform nonmetabolic functions depending on their subcellular location. ATP citrate lyase (ACLY) (a) and the pyruvate dehydrogenase complex (PDC) (b) contribute to histone acetylation, in turn regulating gene expression. (c) The glycolytic enzyme pyruvate kinase M2 (PKM2) activates transcription of glycolytic enzymes when relocated to the nucleus. (d) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as an RNA-binding protein in the absence of glycolytic substrate, dampening translation of cytokines.
cells [50], and autophagy in yeast [51]. Interestingly, the pyruvate dehydrogenase complex (PDC), originally located only in mitochondria [52,53], was recently found to translocate to the nucleus where it promotes the production of acetyl-CoA from pyruvate (Figure 3) [54]. PDC’s nuclear activity has been suggested to regulate transcription of genes involved in cell cycle progression through histone acetylation [54]. A nonmetabolic function has also been described for the glycolytic pyruvate kinase M2 (PKM2). PKM2 translocates from the cytoplasm to the nucleus, where it acts as a transcriptional coactivator (Figure 3) [55–57]. Nuclear PKM2 induces the transcription of glycolysis-related genes, correlating with cancer cell proliferation and tumorigenesis [56,58]. Interestingly, nuclear localization of the enzyme is promoted by acetylation of a specific lysine residue in PKM2 [59]. In cancer cells and macrophages, nuclear PKM2 interacts with HIF-1a and thereby regulates the expression of glycolytic enzymes (in both cell types) and pro-inflammatory cytokines (in macrophages) [56,60]. In the cytosol, metabolic enzymes can also control translation of specific proteins, like cytokines. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase www.sciencedirect.com
(GAPDH) has been linked to different cellular processes depending on its subcellular localization [61]. In nutrientreplete conditions, this enzyme supports glycolysis in activated T-lymphocytes [62]. However, upon glucose deprivation, GAPDH is not supporting glycolysis for ATP production, but by binding to the 30 -untranslated mRNA of IFN-g, GAPDH dampens translation of this cytokine (Figure 3) [62]. Thus, in the absence of its substrate, GAPDH switches from its role as metabolic enzyme to an RNA-binding protein, thereby providing a metabolic checkpoint for T cells to couple effector status with glycolysis when sufficient nutrient is available. The atypical roles that metabolic enzymes can play depending on their localization emphasize the need to expand our understanding of their specific contributions to cellular functions. These examples suggest that metabolic enzymes act as sensors bridging the gap between nutrient availability and epigenetic modifications, a regulatory mechanism that has already been linked to pathology (e.g. cancer) [63]. Likewise, nutrient availability might drive deregulated metabolic enzyme compartmentalization and activity to alter cellular functions during disease onset and progression. Although speculative, such pioneering insights may provide new targets for existing therapeutic strategies. Current Opinion in Biotechnology 2015, 34:73–81
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Temporal cell cycle-dependent metabolic compartmentalization The spatial compartmentalization of metabolic pathways discussed so far incompletely describes the integration between metabolic organization and cellular function. A more comprehensive picture emerges when time-related/ temporal compartmentalization is taken into account. A clear example of how temporal compartmentalization affects cellular function is the dynamic regulation of cell cycle progression (Figure 4) [64,65]. Proliferating cells must transiently adapt their metabolism to generate sufficient energy and metabolic intermediates to meet the anabolic demands of biomass production. The progression throughout the cell cycle itself requires high levels of glucose consumption. More in particular, the glycolytic machinery is engaged during the transition from quiescence to proliferation [66,67]. During the G1-S transition, the APC/C-Cdh1 ubiquitin ligase system negatively regulates the expression of PFKFB3 and glutaminase [68,69]. Therefore, the inactivation of the APC/ Cdh1 complex marks the entry of cells to the S phase and the concomitant supporting increase in glycolysis and glutaminolysis [70,71]. Moreover, the pentose phosphate pathway (PPP), which produces an essential precursor of nucleotide synthesis (ribose-5-phosphate), is enhanced during late G1 and S phase [72]. Consistently, the expression of the rate-limiting enzyme of the PPP (glucose-6-phosphate dehydrogenase) and nucleotide metabolism (phosphoribosyl pyrophosphate synthetase) is increased in proliferating cells [73,74]. The increase in glucose metabolism is remarkable but by itself not sufficient to sustain proliferation. The involvement of mitochondria during DNA synthesis has also been suggested, and suppressing oxidative metabolism or lipogenesis stalls proliferation in certain cancer Figure 4
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Metabolic regulation during cell cycle progression. Schematic representation of the relationship between the phase of the cell cycle and the expression levels of the glycolytic enzyme PFKFB3 and the glutaminolytic enzyme GLS1. Current Opinion in Biotechnology 2015, 34:73–81
cells [75,76]. Many mechanisms of temporal metabolic compartmentalization remain to be elucidated and this will not only provide novel insights into how metabolic rewiring support proliferation and related processes such as cell differentiation, but might also offer new approaches to target proliferative diseases.
Conclusions and perspectives Although the spatial and temporal dynamics of metabolism are becoming clearer, the precise implications of compartmentalized energy production, biosynthesis and redox homeostasis in cellular function remain blurred. Indeed, metabolic compartmentalization studies have been hampered by the lack of adequate metabolite tracing methods. An example of how new technical developments can improve our understanding of the specific role of compartmentalized metabolic pathways was recently provided. Using new methods that trace deuterium into NADPH, two studies determined the direction of the serine/glycine pathways in mitochondria versus the cytosol, shedding light on the relative amounts of NADPH produced and its eventual contribution to distinct cellular processes [77,78,79]. Understanding metabolic compartmentalization can also offer novel therapeutic opportunities. For instance, the finding that glycolytic enzymes compartmentalize to lamellipodia of migrating endothelial cells implicates glycolysis as an appealing anti-angiogenic target [6,66,80]. The distinct microenvironments in different cellular compartments (pH, redox potential, etc.) offer opportunities for new strategies to activate drugs in specific compartments or guide their delivery to particular subcellular compartments [81,82]. An example includes a mitochondrion-targeted dichloroacetate (MitoDCA) variant that indirectly stimulates pyruvate dehydrogenase (PDH) and induces cancer cell apoptosis [83]. Because of differences in mitochondrial and plasma membrane potential, this compound selectively targets mitochondria, and induces cancer cell apoptosis more efficiently than DCA alone. An intriguing question is whether the compartmentalization of PDH (one enzyme of the PDC previously discussed) in the nucleus to alter histone acetylation [54] might represent a mechanism of cancer cell resistance to DCA. Altogether, the examples discussed in this perspective highlight that understanding compartmentalization of metabolic pathways not only might be critical to understand the fundamental basics of cellular metabolism, but might also offer unexplored therapeutic opportunities.
Acknowledgements We apologize for not being able to cite the work of all other studies related to this topic because of space restrictions. P.S. is a postdoctoral fellow supported by the Belgian American Educational Foundation (BAEF). J.G. is a PhD student supported by a fellowship from the FWO. The work of P.C. is supported by a Federal Government Belgium grant (IUAP P7/03), www.sciencedirect.com
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long-term structural Methusalem funding by the Flemish Government, the Belgian Science Fund (FWO grants; G.0817.11, 0578.12, 0834.13), the Foundation of Leducq Transatlantic Artemic Network, a European Research Council (ERC) Advanced Research Grant and the AXA Research Fund.
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39. Sheng ZH, Cai Q: Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nat Rev Neurosci 2012, 13:77-93. 40. Ivannikov MV, Sugimori M, Llinas RR: Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume. J Mol Neurosci 2013, 49:223-230. 41. Spillane M, Ketschek A, Merianda TT, Twiss JL, Gallo G: Mitochondria coordinate sites of axon branching through localized intra-axonal protein synthesis. Cell Rep 2013, 5:1564-1575. An unexpected role for mitochondria in protein translation is uncovered in this study. Branching of sensory axons is dependent on the relocation of mitochondria at hot spots of mRNA translation, where the organelles respire and produce ATP. 42. Zala D, Hinckelmann MV, Yu H, Lyra da Cunha MM, Liot G, Cordelieres FP, Marco S, Saudou F: Vesicular glycolysis provides on-board energy for fast axonal transport. Cell 2013, 152:479-491. Glycolytic enzymes are shown to be involved in fast axonal transport by providing ATP necessary to fuel vesicle movement over long distances in axons. 43. Ivannikov MV, Sugimori M, Llinas RR: Calcium clearance and its energy requirements in cerebellar neurons. Cell Calcium 2010, 47:507-513. 44. Wu K, Aoki C, Elste A, Rogalski-Wilk AA, Siekevitz P: The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide. Proc Natl Acad Sci U S A 1997, 94:13273-13278. 45. Hertz L, Peng L, Dienel GA: Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J Cereb Blood Flow Metab 2007, 27:219-249. 46. Belanger M, Allaman I, Magistretti PJ: Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab 2011, 14:724-738. 47. Cai L, Sutter BM, Li B, Tu BP: Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes. Mol Cell 2011, 42:426-437. 48. Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB: ATP-citrate lyase links cellular metabolism to histone acetylation. Science 2009, 324:1076-1080. 49. Hatzivassiliou G, Zhao F, Bauer DE, Andreadis C, Shaw AN, Dhanak D, Hingorani SR, Tuveson DA, Thompson CB: ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 2005, 8:311-321. 50. Lee JV, Carrer A, Shah S, Snyder NW, Wei S, Venneti S, Worth AJ, Yuan ZF, Lim HW, Liu S et al.: Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab 2014, 20(2):306-319. This study confirms the role of the nuclear-compartmentalized ATP citrate lyase in the regulation of histone acetylation in cancer cells. Of note, acetyl-CoA metabolism is implicated in the control of cancer cell epigenome. 51. Eisenberg T, Schroeder S, Andryushkova A, Pendl T, Kuttner V, Bhukel A, Marino G, Pietrocola F, Harger A, Zimmermann A et al.: Nucleocytosolic depletion of the energy metabolite acetylcoenzyme a stimulates autophagy and prolongs lifespan. Cell Metab 2014, 19:431-444. 52. Hoshi M, Takashima A, Noguchi K, Murayama M, Sato M, Kondo S, Saitoh Y, Ishiguro K, Hoshino T, Imahori K: Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain. Proc Natl Acad Sci U S A 1996, 93:2719-2723. Current Opinion in Biotechnology 2015, 34:73–81
55. Gao X, Wang H, Yang JJ, Liu X, Liu ZR: Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell 2012, 45:598-609. 56. Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A, Semenza GL: Pyruvate kinase M2 is a PHD3stimulated coactivator for hypoxia-inducible factor 1. Cell 2011, 145:732-744. 57. Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, Gao X, Aldape K, Lu Z: Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 2011, 480:118-122. 58. Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC, Lu Z: ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol 2012, 14:1295-1304. This work uncover the role of PKM2 as a transcriptional activator. The translocation of the enzyme to the nucleus is required for upregulation of genes involved in the Warburg effect. 59. Lv L, Xu YP, Zhao D, Li FL, Wang W, Sasaki N, Jiang Y, Zhou X, Li TT, Guan KL et al.: Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell 2013, 52:340-352. 60. Yang L, Xie M, Yang M, Yu Y, Zhu S, Hou W, Kang R, Lotze MT, Billiar TR, Wang H et al.: PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis. Nat Commun 2014, 5:4436. 61. Tristan C, Shahani N, Sedlak TW, Sawa A: The diverse functions of GAPDH: views from different subcellular compartments. Cell Signal 2011, 23:317-323. 62. Chang CH, Curtis JD, Maggi LB Jr, Faubert B, Villarino AV, O’Sullivan D, Huang SC, van der Windt GJ, Blagih J, Qiu J et al.: Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell 2013, 153:1239-1251. This study addresses the outstanding question of whether aerobic glycolysis plays a role in T cell activation. Under glucose depleted conditions, the glycolytic enzyme GAPDH switches to an mRNA-binding protein to control cytokine expression. 63. Dawson MA, Kouzarides T: Cancer epigenetics: from mechanism to therapy. Cell 2012, 150:12-27. 64. Bertoli C, Skotheim JM, de Bruin RA: Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 2013, 14:518-528. 65. Lunt SY, Vander Heiden MG: Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol 2011, 27:441-464. 66. Schoors S, De Bock K, Cantelmo AR, Georgiadou M, Ghesquiere B, Cauwenberghs S, Kuchnio A, Wong BW, Quaegebeur A, Goveia J et al.: Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Cell Metab 2014, 19:37-48. This report provides a pharmacological proof-of-principle on the role of PFKFB3 in vessel sprouting. It demonstrates the efficacy of inhibition of the glycolytic pathway in pathological angiogenesis. 67. Wang T, Marquardt C, Foker J: Aerobic glycolysis during lymphocyte proliferation. Nature 1976, 261:702-705. 68. Almeida A, Bolanos JP, Moncada S: E3 ubiquitin ligase APC/CCdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation. Proc Natl Acad Sci U S A 2010, 107:738-741. www.sciencedirect.com
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69. Colombo SL, Palacios-Callender M, Frakich N, Carcamo S, Kovacs I, Tudzarova S, Moncada S: Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells. Proc Natl Acad Sci U S A 2011, 108:21069-21074.
77. Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB, Rabinowitz JD: Quantitative flux analysis reveals folatedependent NADPH production. Nature 2014, 510:298-302. Using a novel method for metabolic flux analysis, this work demonstrated the role of the serine/glycine metabolism in the generation of NADPH.
70. Colombo SL, Palacios-Callender M, Frakich N, De Leon J, Schmitt CA, Boorn L, Davis N, Moncada S: Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes. Proc Natl Acad Sci U S A 2010, 107:18868-18873.
78. Lewis CA, Parker SJ, Fiske BP, McCloskey D, Gui DY, Green CR, Vokes NI, Feist AM, Vander Heiden MG, Metallo CM: Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell 2014, 55:253-263. This work illustrates the directionality of the serine/glycine metabolism reactions occurring in both the cytoplasm and mitochondria by using innovative techniques.
71. Tudzarova S, Colombo SL, Stoeber K, Carcamo S, Williams GH, Moncada S: Two ubiquitin ligases, APC/C-Cdh1 and SKP1CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle. Proc Natl Acad Sci U S A 2011, 108:5278-5283. 72. Vizan P, Alcarraz-Vizan G, Diaz-Moralli S, Solovjeva ON, Frederiks WM, Cascante M: Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29. Int J Cancer 2009, 124:2789-2796. 73. Du W, Jiang P, Mancuso A, Stonestrom A, Brewer MD, Minn AJ, Mak TW, Wu M, Yang X: TAp73 enhances the pentose phosphate pathway and supports cell proliferation. Nat Cell Biol 2013, 15:991-1000. These findings establish a role for the pentose phosphate pathways enzyme G6PD in providing NAPDH and ribose necessary for the progression through the cell cycle. 74. Hovi T, Allison AC, Allsop J: Rapid increase of phosphoribosyl pyrophosphate concentration after mitogenic stimulation of lymphocytes. FEBS Lett 1975, 55:291-293. 75. Kuhajda FP: Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 2000, 16:202-208. 76. Lupu R, Menendez JA: Pharmacological inhibitors of Fatty Acid Synthase (FASN)-catalyzed endogenous fatty acid biogenesis: a new family of anti-cancer agents? Curr Pharm Biotechnol 2006, 7:483-493.
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79. Labuschagne CF, van den Broek NJ, Mackay GM, Vousden KH, Maddocks OD: Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells. Cell Rep 2014, 7:1248-1258. 80. Schoors S, Cantelmo AR, Georgiadou M, Stapor P, Wang X, Quaegebeur A, Cauwenberghs S, Wong BW, Bifari F, Decimo I et al.: Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy? Cell Cycle 2014, 13:16-22. This publication further profiled the efficacy and safety of pharmacological inhibition of glycolysis and PFKFB3 as an anti-angiogenic theraphy. 81. Dhar S, Lippard SJ: Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate. Proc Natl Acad Sci U S A 2009, 106:22199-22204. 82. Ulbrich K, Subr V: Polymeric anticancer drugs with pHcontrolled activation. Adv Drug Deliv Rev 2004, 56:1023-1050. 83. Pathak RK, Marrache S, Harn DA, Dhar S: Mito-DCA: a mitochondria targeted molecular scaffold for efficacious delivery of metabolic modulator dichloroacetate. ACS Chem Biol 2014, 9:1178-1187. 84. Shiraishi T, Verdone JE, Huang J, Kahlert UD, Hernandez JR, Torga G, Zarif JC, Epstein T, Gatenby R, McCartney A et al.: Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells. Oncotarget 2014. In Press.
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ScienceDirect Metabolic pathway compartmentalization: an underappreciated opportunity? Annalisa Zecchin1,2, Peter C Stapor1,2, Jermaine Goveia1,2 and Peter Carmeliet1,2 For eukaryotic cells to function properly, they divide their intracellular space in subcellular compartments, each harboring specific metabolic activities. In recent years, it has become increasingly clear that compartmentalization of metabolic pathways is a prerequisite for certain cellular functions. This has for instance been documented for cellular migration, which relies on subcellular localization of glycolysis or mitochondrial respiration in a cell type-dependent manner. Although exciting, this field is still in its infancy, partly due to the limited availability of methods to study the directionality of metabolic pathways and to visualize metabolic processes in distinct cellular compartments. Nonetheless, advances in this field may offer opportunities for innovative strategies to target deregulated compartmentalized metabolism in disease. Addresses 1 Laboratory of Angiogenesis & Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium 2 Laboratory of Angiogenesis & Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium
metabolic pathways are compartmentalized in time and space to support specific cellular processes. The realization that metabolism controls cell function in health and disease has lead to a resurgent interest in cellular metabolism, with the ultimate goal of targeting metabolism therapeutically. However, it is clear that a better understanding of how metabolic compartmentalization contributes to cell function could help to identify novel, perhaps even more specific, therapeutic approaches. We will not provide an all-encompassing literature survey, but rather discuss representative examples that highlight the importance of metabolic compartmentalization to support cell function.
How and why is metabolism compartmentalized?
Introduction
Physical delimitation of metabolic pathways within membrane-delineated organelles or the cytosol is a classical example of metabolic compartmentalization [3]. Metabolic pathways can be localized in distinct domains, even within one compartment. For instance, in smooth muscle cells, glycogen stores are metabolized via oxidative phosphorylation, while external glucose is used for glycolysis [4]. Metabolic enzymes can be also sequestered to scaffold proteins, as demonstrated by the association of glycolytic enzymes with the actin cytoskeleton [5,6,7–9]. Another feature of metabolic enzymes is their ability to assemble in large quaternary structures, ranging from homodimers (fatty acid synthase) and polymers (glutamine synthetase) to multiprotein metabolon complexes (purinosome) [10–13]. Spatial organization of enzyme complexes facilitates transfer of metabolites from one enzyme to the next, thus generating an efficient assembly line and increasing overall metabolic output [5,14,15]. In addition to spatial compartmentalization, metabolic pathways are also temporally compartmentalized. A prime example is the transient peak in glycolysis and glutaminolysis during the S phase of the cell cycle [16].
Given the complexity of biological systems in multicellular organisms, evolution has organized living matter into structured compartments. At the macroscopic level, the human body is divided in distinct organs and tissues. Organs are composed of multiple cell types, cells are divided into organelles, and organelles in turn are defined by spatial architecture. At the molecular level, cellular metabolism is a prime example of an organized complex system. Scientific reductionism has lead to increasingly better descriptions of cellular metabolic networks [1]. However, cells are not simple bags of enzymes and metabolites with random spatial and temporal organization [2]. On the contrary,
There are many benefits to subcellular compartmentalization. First, subcellular compartmentalized microenvironments favor optimal activity of specific enzymes. For instance, peroxisomal oxidase and catalase activity are higher at alkaline pH [17], as is the case in peroxisomes [18,19]. Second, some metabolic reactions produce toxic intermediates that can be contained and disposed of in closed compartments. The containment of hydrogen peroxide and its breakdown by catalase inside peroxisomes is a prime example [20]. Third, compartmentalization can limit diffusion and prevent escape of intermediates
Corresponding author: Carmeliet, Peter ([email protected])
Current Opinion in Biotechnology 2015, 34:73–81 This review comes from a themed issue on Systems biology Edited by Sarah Maria Fendt and Costas D Maranas
http://dx.doi.org/10.1016/j.copbio.2014.11.022 0958-1669/# 2014 Elsevier Ltd. All rights reserved.
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through organelle membranes as is the case for ATP and ADP in muscle cells, which therefore need adenine nucleotide translocase to cross the inner mitochondrial membrane [21]. Fourth, compartmentalization counteracts futile cycles of metabolites that might otherwise be continuously interconverted in reactions operating in opposite directions. In yeast, differential expression of metabolic enzymes periodically occurs over time. Such a precise orchestration regulates energy production through activation of distinct metabolic pathways in restricted temporal windows [22]. Finally, metabolic compartmentalization often increases the efficiency of chemical reactions, as many reactions in free solution would be otherwise diffusion-limited due to restricted local enzyme substrate availability. In brain and muscle, glycogen and glycogenolytic enzymes are compartmentalized together in microdomains to facilitate glycogen breakdown [23]. In the smooth muscle cell example, the internal glycogen stores that are metabolized via oxidative phosphorylation supply energy for actin–myosin contractions, while the external glucose supplies energy for ion transport via glycolysis [24]. Despite the many benefits and obvious necessity of metabolic compartmentalization, its functional role in supporting biological processes is underappreciated. In the remainder of this review, we will provide examples of how metabolic compartmentalization controls specific cell functions.
Meeting energy demand by metabolic pathway compartmentation An example illustrating how metabolic pathways influence biological processes is the hydra, a primitive animal in which distinct metabolic pathways are selectively preferred depending on the type of locomotion adopted. The slow movement of its body over the sea floor relies on oxidative metabolism, while glycolysis is required for the sudden and dynamic changing movements of its tentacles for praying [25]. Likewise, an association of glycolytic enzymes with structures for rapid motility has been described in the tail of sperm cells [26]. Because glycolysis has the capacity for quick ATP production relative to oxidative phosphorylation, it is used for activities requiring high, local levels of ATP over a short period, whereas oxidative phosphorylation more efficiently produces ATP over time. Compartmentalization of glycolysis during cell motility
Cellular migration relies on dynamic remodeling of the cytoskeleton, a process requiring large amounts of local ATP [27,28]. Interaction of glycolytic enzymes with the actin cytoskeleton has been documented in mammalian neurons and skeletal muscle cells as well as in fruitflies and fish muscles. Compartmentalization of glycolysis on the actin cytoskeleton enables efficient and high local ATP production [5,7–9]. The importance of this association is illustrated by the fact that, despite expression of the full complement of active glycolytic enzymes in flight muscles, transgenic fruitflies are unable to fly when Current Opinion in Biotechnology 2015, 34:73–81
glycolytic enzymes fail to colocalize in actin-rich sarcomeres [9]. Migrating endothelial cells rely on highly dynamic actin architectures at the leading edge, lamellipodia and filopodia, which pull the cell body forward [29]. In quiescent endothelial cells, mitochondria and glycolytic enzymes are present in the perinuclear cytosol [6]. In contrast, when these cells start to migrate, they relocate their glycolytic machinery to the lamellipodia and filopodia (Figure 1) [6]. Notably, glycolytic enzymes physically interact and coconcentrate with actin fibers at the membrane ruffles in the lamellipodia, and even become superactivated by this actin association [6]. Interestingly, endothelial cell lamellipodia and filopodia are too narrow and too thin to accommodate bulky mitochondria [6]. Local glycolysis allows rapid generation of ATP for dynamic motility, and prevents taxing ATP demands throughout the cell [6]. Suppressing glycolysis reduces endothelial cell motility, and lamellipodia and filopodia formation, whereas blocking mitochondrial respiration does not affect endothelial cell migration [6]. Similar to ECs, cancer cells often have a high glycolytic flux, which has been implicated in cancer cell invasion. Invasive cancer cells migrate through extracellular matrix using protrusive structures called invadopodia [30]. Proteomic analysis of invadopodia revealed that they are enriched in glycolytic enzymes [31]. High glycolytic flux has accordingly been associated with mesenchymal cancer cell motility and cytoskeleton remodeling, possibly accounting for a more aggressive phenotype [84]. Furthermore, reducing glycolysis in cancer cells decreases their ability to degrade the matrix [31,32]. Mitochondrial compartmentalization during cell motility
Mitochondria are also important for migration of various cancer cells, though this requirement is dependent on the cancer cell type. For instance, invadopodia in human glioma contain active mitochondria [33]. In migrating epithelial cancer cells, mitochondria reside at the leading front, and altering their anterior position impairs migration [34]. In metastatic breast cancer cells, mitochondrial fragmentation promotes, while mitochondrial fusion reduces lamellipodia formation and cell migration [35]. Consistently, inhibition of mitochondrial ATP production reduces migration and actin polymerization, implicating mitochondria as the source of ATP for actin remodeling [35]. Lymphocytes exhibit a profoundly different mode of motility compared to endothelial and cancer cells. In order to migrate in a directional manner, they undergo polarization and generate specialized cell domains. They form a lamellipodium at the leading edge, containing the actin polymerization machinery and chemoattractant receptors, and a uropod at the trailing edge, concentrating adhesion receptors and the microtubule-organizing center (MTOC) www.sciencedirect.com
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Metabolic compartmentalization during cell motility. (a) Glycolytic ATP production resides throughout the perinuclear cytosol in quiescent, nonmotile ECs (left). In angiogenic ECs (right), glycolytic enzymes compartmentalize in lamellipodia and filopodia at the leading front to generate high levels of ATP for actin polymerization. (b) In unpolarized, nonmotile lymphocytes, mitochondria are distributed at the contact zone of adhesion on substratum (left). During directional movement, mitochondria relocalize to the trailing uropod to supply ATP for actomyosin contraction.
that facilitate migration [36]. This change in cell shape enables them to convert cytoskeletal forces into net cell body displacement. At the uropod, myosin filaments contract actin filaments to provide the tension required for migration [36]. In nonmotile lymphocytes, mitochondria are present at the plasma membrane, close to the contact site between activator and effector cells [37]. When lymphocytes are migrating, mitochondria are transported to the uropod alongside microtubules in a process requiring mitochondrial fission (Figure 1). This strategic position allows mitochondria to supply sufficient local amounts of ATP for the high energy-demanding actomyosin contractions, thereby enabling uropod retraction and cell displacement [38]. The role of glycolysis compartmentalization in lymphocyte migration has not been studied yet. Thus, www.sciencedirect.com
endothelial, cancer and immune cells all differ in their metabolic requirements and compartmentalization during migration. Understanding the differences in compartmentalized metabolism across various cell types (e.g. endothelial versus cancer cells) may provide advantages for designing therapies to target selected cell types. Compartmentalized ATP generation in sustaining neural function
Cells have not only capitalized on the different glycolytic and mitochondrial ATP production dynamics in supporting cytoskeletal dynamics but also in many other processes. For example, compartmentalization of metabolism in neurons is required for synaptic transmission and intracellular transport. Synaptic transmission consumes large Current Opinion in Biotechnology 2015, 34:73–81
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amounts of ATP for synchronized vesicle release, vesicle recycling and generation of synapse potentials [39]. Accordingly, localization of mitochondria to synaptic terminals provides resident hubs for the synthesis of ATP required for synaptic transmission (Figure 2) [40]. Production of ATP by mitochondria is also critical for axon branching of sensory neurons. In neurons, the machinery for protein translation that is necessary for actin-dependent branching is located at axon branching points, but mRNA translation that promotes branching occurs only at axonal locations where mitochondria stall and produce ATP (Figure 2) [41].
demonstrate that neurons compartmentalize different metabolic pathways for specific vesicle related functions, it will be interesting to determine whether this is also true in other cell types and whether other vesicular processes, like endocytosis, are supported by compartmentalized metabolism.
Location-dependent functions of metabolic enzymes Besides being essential for energy production, macromolecular biosynthesis and redox homeostasis in the cytosol or mitochondria, metabolic enzymes can have signaling roles in the nucleus. For instance, emerging evidence supports a link between metabolism and gene transcription. Indeed, levels of acetyl-CoA, the substrate of histone acetyl-transferases, regulate the expression of genes through histone acetylation [47,48]. While cytosolic ATP-citrate lyase (ACLY) is critical for the conversion of citrate to acetyl-CoA, necessary for lipogenesis [49], nuclear localization of ACLY suggests a role in the production of acetyl-CoA in the nucleus, where it can be used for histone acetylation (Figure 3) [48]. Linking nutrient availability to production of acetyl-CoA, ACLY has been correlated to the transcription of genes involved in adipocyte differentiation [48], proliferation in cancer
In contrast, glycolysis provides fuel for fast axonal transport of vesicles to nerve terminals (Figure 2) [42]. Vesicles, which traffic along microtubules and carry trophic factors, possess glycolytic machinery to generate their own ATP to fuel dynein and kinesin transport motors [42]. In addition, glycolysis is compartmentalized at the plasma membrane to fuel high energydemanding ion pumps in neurons [43,44], and in filopodia of astrocytes, glycogen stores have been identified in filopodia-like cellular protrusions [45], likely to release lactate in the extracellular space for oxidative metabolism by neighboring neurons [46]. Although these studies Figure 2
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Metabolic compartmentalization in neural functions. Glycolytic enzymes associate with transported vesicles to provide ATP for motor proteins (a). Local mitochondrial ATP production supports synaptic vesicle release (b) and mRNA translation at axonal branch points (c). Current Opinion in Biotechnology 2015, 34:73–81
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Location-dependent functions of metabolic enzymes. Metabolic enzymes can perform nonmetabolic functions depending on their subcellular location. ATP citrate lyase (ACLY) (a) and the pyruvate dehydrogenase complex (PDC) (b) contribute to histone acetylation, in turn regulating gene expression. (c) The glycolytic enzyme pyruvate kinase M2 (PKM2) activates transcription of glycolytic enzymes when relocated to the nucleus. (d) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as an RNA-binding protein in the absence of glycolytic substrate, dampening translation of cytokines.
cells [50], and autophagy in yeast [51]. Interestingly, the pyruvate dehydrogenase complex (PDC), originally located only in mitochondria [52,53], was recently found to translocate to the nucleus where it promotes the production of acetyl-CoA from pyruvate (Figure 3) [54]. PDC’s nuclear activity has been suggested to regulate transcription of genes involved in cell cycle progression through histone acetylation [54]. A nonmetabolic function has also been described for the glycolytic pyruvate kinase M2 (PKM2). PKM2 translocates from the cytoplasm to the nucleus, where it acts as a transcriptional coactivator (Figure 3) [55–57]. Nuclear PKM2 induces the transcription of glycolysis-related genes, correlating with cancer cell proliferation and tumorigenesis [56,58]. Interestingly, nuclear localization of the enzyme is promoted by acetylation of a specific lysine residue in PKM2 [59]. In cancer cells and macrophages, nuclear PKM2 interacts with HIF-1a and thereby regulates the expression of glycolytic enzymes (in both cell types) and pro-inflammatory cytokines (in macrophages) [56,60]. In the cytosol, metabolic enzymes can also control translation of specific proteins, like cytokines. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase www.sciencedirect.com
(GAPDH) has been linked to different cellular processes depending on its subcellular localization [61]. In nutrientreplete conditions, this enzyme supports glycolysis in activated T-lymphocytes [62]. However, upon glucose deprivation, GAPDH is not supporting glycolysis for ATP production, but by binding to the 30 -untranslated mRNA of IFN-g, GAPDH dampens translation of this cytokine (Figure 3) [62]. Thus, in the absence of its substrate, GAPDH switches from its role as metabolic enzyme to an RNA-binding protein, thereby providing a metabolic checkpoint for T cells to couple effector status with glycolysis when sufficient nutrient is available. The atypical roles that metabolic enzymes can play depending on their localization emphasize the need to expand our understanding of their specific contributions to cellular functions. These examples suggest that metabolic enzymes act as sensors bridging the gap between nutrient availability and epigenetic modifications, a regulatory mechanism that has already been linked to pathology (e.g. cancer) [63]. Likewise, nutrient availability might drive deregulated metabolic enzyme compartmentalization and activity to alter cellular functions during disease onset and progression. Although speculative, such pioneering insights may provide new targets for existing therapeutic strategies. Current Opinion in Biotechnology 2015, 34:73–81
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Temporal cell cycle-dependent metabolic compartmentalization The spatial compartmentalization of metabolic pathways discussed so far incompletely describes the integration between metabolic organization and cellular function. A more comprehensive picture emerges when time-related/ temporal compartmentalization is taken into account. A clear example of how temporal compartmentalization affects cellular function is the dynamic regulation of cell cycle progression (Figure 4) [64,65]. Proliferating cells must transiently adapt their metabolism to generate sufficient energy and metabolic intermediates to meet the anabolic demands of biomass production. The progression throughout the cell cycle itself requires high levels of glucose consumption. More in particular, the glycolytic machinery is engaged during the transition from quiescence to proliferation [66,67]. During the G1-S transition, the APC/C-Cdh1 ubiquitin ligase system negatively regulates the expression of PFKFB3 and glutaminase [68,69]. Therefore, the inactivation of the APC/ Cdh1 complex marks the entry of cells to the S phase and the concomitant supporting increase in glycolysis and glutaminolysis [70,71]. Moreover, the pentose phosphate pathway (PPP), which produces an essential precursor of nucleotide synthesis (ribose-5-phosphate), is enhanced during late G1 and S phase [72]. Consistently, the expression of the rate-limiting enzyme of the PPP (glucose-6-phosphate dehydrogenase) and nucleotide metabolism (phosphoribosyl pyrophosphate synthetase) is increased in proliferating cells [73,74]. The increase in glucose metabolism is remarkable but by itself not sufficient to sustain proliferation. The involvement of mitochondria during DNA synthesis has also been suggested, and suppressing oxidative metabolism or lipogenesis stalls proliferation in certain cancer Figure 4
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Metabolic regulation during cell cycle progression. Schematic representation of the relationship between the phase of the cell cycle and the expression levels of the glycolytic enzyme PFKFB3 and the glutaminolytic enzyme GLS1. Current Opinion in Biotechnology 2015, 34:73–81
cells [75,76]. Many mechanisms of temporal metabolic compartmentalization remain to be elucidated and this will not only provide novel insights into how metabolic rewiring support proliferation and related processes such as cell differentiation, but might also offer new approaches to target proliferative diseases.
Conclusions and perspectives Although the spatial and temporal dynamics of metabolism are becoming clearer, the precise implications of compartmentalized energy production, biosynthesis and redox homeostasis in cellular function remain blurred. Indeed, metabolic compartmentalization studies have been hampered by the lack of adequate metabolite tracing methods. An example of how new technical developments can improve our understanding of the specific role of compartmentalized metabolic pathways was recently provided. Using new methods that trace deuterium into NADPH, two studies determined the direction of the serine/glycine pathways in mitochondria versus the cytosol, shedding light on the relative amounts of NADPH produced and its eventual contribution to distinct cellular processes [77,78,79]. Understanding metabolic compartmentalization can also offer novel therapeutic opportunities. For instance, the finding that glycolytic enzymes compartmentalize to lamellipodia of migrating endothelial cells implicates glycolysis as an appealing anti-angiogenic target [6,66,80]. The distinct microenvironments in different cellular compartments (pH, redox potential, etc.) offer opportunities for new strategies to activate drugs in specific compartments or guide their delivery to particular subcellular compartments [81,82]. An example includes a mitochondrion-targeted dichloroacetate (MitoDCA) variant that indirectly stimulates pyruvate dehydrogenase (PDH) and induces cancer cell apoptosis [83]. Because of differences in mitochondrial and plasma membrane potential, this compound selectively targets mitochondria, and induces cancer cell apoptosis more efficiently than DCA alone. An intriguing question is whether the compartmentalization of PDH (one enzyme of the PDC previously discussed) in the nucleus to alter histone acetylation [54] might represent a mechanism of cancer cell resistance to DCA. Altogether, the examples discussed in this perspective highlight that understanding compartmentalization of metabolic pathways not only might be critical to understand the fundamental basics of cellular metabolism, but might also offer unexplored therapeutic opportunities.
Acknowledgements We apologize for not being able to cite the work of all other studies related to this topic because of space restrictions. P.S. is a postdoctoral fellow supported by the Belgian American Educational Foundation (BAEF). J.G. is a PhD student supported by a fellowship from the FWO. The work of P.C. is supported by a Federal Government Belgium grant (IUAP P7/03), www.sciencedirect.com
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long-term structural Methusalem funding by the Flemish Government, the Belgian Science Fund (FWO grants; G.0817.11, 0578.12, 0834.13), the Foundation of Leducq Transatlantic Artemic Network, a European Research Council (ERC) Advanced Research Grant and the AXA Research Fund.
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