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G-protein-coupled receptor regulation of de novo purine biosynthesis: a novel druggable mechanism a

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Ye Fang , Jarrod French , Hong Zhao & Stephen Benkovic

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Biochemical Technologies, Science and Technology Division , Corning Incorporated , Corning , New York , USA b

Department of Chemistry , Pennsylvania State University , University Park , PA , USA Published online: 24 Jul 2013.

To cite this article: Ye Fang , Jarrod French , Hong Zhao & Stephen Benkovic (2013) G-proteincoupled receptor regulation of de novo purine biosynthesis: a novel druggable mechanism, Biotechnology and Genetic Engineering Reviews, 29:1, 31-48, DOI: 10.1080/02648725.2013.801237 To link to this article: http://dx.doi.org/10.1080/02648725.2013.801237

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Biotechnology and Genetic Engineering Reviews, 2013 Vol. 29, No. 1, 31–48, http://dx.doi.org/10.1080/02648725.2013.801237

G-protein-coupled receptor regulation of de novo purine biosynthesis: a novel druggable mechanism Ye Fanga*, Jarrod Frenchb, Hong Zhaob and Stephen Benkovicb* a Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York, USA; bDepartment of Chemistry, Pennsylvania State University, University Park, PA, USA

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(Received 29 September 2012; accepted 31 March 2013) Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery. Keywords: drug discovery; dynamic mass redistribution assay; G protein-coupled receptor; metabolic flux; mitogenic signaling; purine de novo biosynthesis; purine salvage pathway; purinosome

Introduction The molecular architectures of intracellular signaling and metabolic networks have been increasingly recognized to be important in regulating cell function and determining the decision process and fate of cells upon environmental changes. Cells have long been documented to contain several well-defined subcellular microstructures such as the nucleus, mitochondrion, endoplasmic reticulum, ribosome and Golgi apparatus. Advances in imaging techniques and molecular genetics have begun to reveal that many macromolecules associate temporally and spatially to form macromolecular assemblies during various signaling and metabolic processes. For instance, exosomes, nanovesicles secreted by a wide range of mammalian cell types, are shown to carry various molecular constituents of their cell of origin, including proteins and RNA (Valadi et al., 2007; Alvarez-Erviti et al., 2011). Cell membrane nanotubes, formed de novo between cells, create complex intercellular networks that facilitate exchange of information via selective transfer of cellular contents between cells (Mittelbrunn & Sánchez-Madrid, 2012; Rustom, Saffrich, Markovic, Walther, & Gerdes, 2004; Chauveau, Aucher, Eissmann, Vivier, & Davis, 2010). Another example is a microcompartment, in which a protein shell surrounds and encloses various enzymes; these cellular bodies are primitive organelles of bacteria and other microbes and are important for efficient carbon fixation and other chemical reactions (Kerfeld et al., 2005). *Corresponding author. Email: [email protected]; [email protected] Ó 2013 Corning Incorporated

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Substrate channeling, the passing of a metabolic intermediate from one enzyme directly to another without its release into solution, is believed to be common in various metabolic pathways including purine de novo biosynthesis (Huang, Holden, & Raushel, 2001). The mechanisms of how substrate channeling occurs in living cells, however, are largely unknown. Purine de novo biosynthesis constitutes 10 enzymatically catalyzed biochemical transformations for the conversion of phosphoribosyl pyrophosphate (PRPP) to inosine 5’-monophosphate (IMP; Kappock, Ealick, & Stubbe, 2006; Zhang, Morar, & Ealick, 2008). Recently, imaging the distribution of fluorescence protein-tagged enzymes involved in this pathway has led to the discovery of purinosomes – multi-enzyme complexes or depots that are formed upon purine depletion and dispersed upon exposure to purine-rich media (An, Kumar, Sheets, & Benkovic, 2008). Subsequent studies have also revealed the molecular composition, organization and regulation of purinosome formation and dissociation (An, Deng, Tomsho, Kyoung, & Benkovic, 2010; An, Kyoung, Allen, Shokat, & Benkovic, 2010; Field, Anderson, & Stover, 2011; Verrier et al., 2011). In particular, the activation of several endogenous Gi-coupled receptors including the α2A-adrenergic receptor has been shown to promote purinosome formation in the cytosol of native HeLa cells under normal culture conditions, suggesting for the first time an alternative route for propagation of G-protein-coupled receptor (GPCR) mitogenic signaling. As a family of versatile signaling proteins and the largest drug target class (Imming, Sinning, & Meyer, 2006), GPCR mitogenic signaling that promotes cell growth is believed to be a result of multiple signal transduction pathways that act together to relay directly the mitogenic signal to the nucleus (Rozengurt, 2007). Given that GPCR signaling has been implicated in many diseases including cancer and inflammatory disorders (Dorsam & Gutkind, 2007; Lappano & Maggiolini, 2011), the GPCR signaling regulated purinosome formation may represent a novel mechanism for developing new therapeutics. Here, we first review nucleotide metabolism and its regulation, and then present the current understanding of the molecular and cellular mechanisms that regulate the purinosome. We also discuss the implications of the discovery of the purinosome for drug discovery. Nucleotide metabolism Nucleotides are critical players in a multitude of cellular processes. Purines and pyrimidines are the building blocks of DNA and RNA molecules. Adenosine triphosphate (ATP) is the central cellular energy supply, while guanosine triphosphate (GTP) and modified nucleotides such as cyclic AMP (cAMP) are signaling molecules. Nucleotides are also incorporated in cofactors such as nicotinamide adenine dinucleotide and coenzyme A, and serve as precursors to glycogen synthesis in forms such as uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-mannose. Nucleotide metabolism includes de novo synthesis, salvage and catabolism of nucleotides. These metabolic pathways ensure net synthesis of nucleotides and control the levels of nucleotide pools. The nucleotide de novo biosynthetic pathways use amino acids and sugar to produce nucleotides, while the salvage pathway degrades precursor macromolecules to recycle nucleotides. The de novo biosynthesis is essential to all other steps in nucleotide metabolism as well as for several other interconnected pathways. Differentiated cells mostly use the salvage pathways to gain access to nucleotides and only synthesize a small amount of nucleotides to meet their need for synthesis of DNA, RNA and enzyme co-factors (Ljungdahl & Daignan-Fornier, 2012). Rapidly dividing

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cells have much higher purine and pyrimidine demands and thus rely heavily upon de novo biosynthetic pathways (Mayer et al., 1990). The purine de novo biosynthetic pathway is nearly ubiquitous among organisms, but not expressed in all cell types in human. It is, however, highly active in muscle and liver (Becker & Kim, 1987). Conversely, human erythrocyte and mature neuron cells are not capable of purine de novo biosynthesis and rely solely on the salvage pathway (Dudzinska, Hlynczak, Skotnicka, & Suska, 2006). The major site of purine synthesis is in the liver, where purine nucleotides are synthesized de novo as the mononucleotide IMP is further converted to adenosine monophosphate (AMP) or guanosine monophosphate (GMP) through two distinct reaction pathways. The conversion to AMP requires energy in the form of GTP, while the pathway to GMP requires energy in the form of ATP, thus allowing the cell to control the proportions of AMP and GMP. IMP can be synthesized through de novo and salvage pathways. The salvage pathway catalyzes the one-step conversion of hypoxanthine to IMP by hypoxanthine phosphoribosyl transferase (HPRT). The de novo biosynthesis involves 10 enzymatic transformations that convert PRPP to IMP (Figure 1). It requires four moles of ATP, two moles of glutamine, one mole of glycine, one mole of CO2, one mole of aspartate and two moles of formate. The formyl moieties are carried on tetrahydrofolate (THF) in the form of N5,N10-methenylTHF and N10-formylTHF. In mammals, the purine de novo biosynthesis is catalyzed by six enzymes in eukaryotes including PRPP amidotransferase (PPAT; EC 2.4.2.14), trifunctional phosphoribosylglycinamide formyltransferase (GAR Tfase, EC 2.1.2.2)/phosphoribosylglycinamide synthetase (GARS, EC 6.3.4.13)/phosphoribosylaminoimidazole synthetase (AIRS, EC 6.3.3.1) (GART or TrifGART), phosphoribosyl formylglycinamidine synthase (EC 6.3.5.3) (FGAMS), bifunctional phosphoribosyl aminoimidazole carboxylase (CAIRS, EC 4.1.1.21)/phosphoribosyl aminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS), adenylosuccinate lyase (EC 4.3.2.2) (ADSL) and bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICAR Tfase, EC 2.1.2.3)/IMP cyclohydrolase (IMPCH, EC 3.5.4.10) (ATIC). Amongst them, PPAT, FGAMS and ADSL are monofunctional enzymes, while PAICS and ATIC are bifunctional enzymes, and TrifGART a trifunctional enzyme. PPAT catalyzes step 1, which is the rate-limiting and regulative step. TrifGART catalyzes steps 2, 3 and 5, while FGAMS catalyzes step 4, PAICS steps 6 and 7, ADSL step 8, and ATIC steps 9 and 10. ADSL also catalyzes the second of the two steps converting IMP to AMP. In mammals the pyrimidine de novo biosynthesis is accomplished by six-step enzymatic transformations to generate uridine monophosphate (UMP; Figure 1). The first step is to generate carbamoyl phosphate from carbonate and the amide nitrogen of glutamine, catalyzed by the carbamoyl phosphate synthetase II activity of the trifunctional cytosolic mammalian enzyme Cad. This step is the rate-limiting step, being feedback inhibited by uridine diphosphate (UDP) and uridine triphosphate (UTP) and activated by PRPP and ATP. The second step is to generate carbamoyl aspartate via the condensation of carbamoyl phosphate with aspartate, catalyzed by the aspartate transcarbamoylase activity of Cad. The third step is to generate dihydroorotate via ring closure of carbamoyl aspartate, catalyzed by the dihydroorotase activity of Cad. The fourth step is to generate orotate via oxidization of dihydroorotate by dihydroorotate dehydrogenase located in the mitochondria, wherein quinones supply the oxidizing power. The fifth step is to form orotidine-5'-monophosphate (OMP) via coupling of orotate with PRPP, catalyzed by the orotate phosphoribosyl transferase activity of the bifunctional enzyme Umps. Lastly, OMP is then decarboxylated to form UMP by the OMP decarboxylase

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Figure 1. Nucleotide de novo biosynthesis network. Note: (Left) The six-step transformations for pyrimidine de novo biosynthesis pathway. (Right) The ten-step transformations for purine de novo biosynthesis pathway. (Middle) The folatemediated one-carbon metabolism in the cytoplasm and nucleus. One-carbon metabolism in the cytoplasm is required for the de novo synthesis of purines and thymidylate. The wide dotted lines are to separate out the three different pathways. PRPP, 5-phosphoribosyl-1-pyrophosphate; PRA, 5-phospho-d-ribosylamine; GAR, glycinamide ribonucleotide; FGAR, N-formylglycinamide ribonucleotide; FGAM, N-formylglycinamidine ribonucleotide; AIR, aminoimidazole ribonucleotide; CAIR, carboxyaminoimidazole ribonucleotide; SAICAR, N-succinocarboxamide5-aminoimidazole ribonucleotide; AICAR, aminoimidazole-4-carboxamide ribonucleotide; FAICAR, 5-formamido-4-imidazolecarboxamide ribonucleotide; IMP, inosine monophosphate; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; TYMS, thymidylate synthase; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADP+, nicotinamide adenine dinucleotide phosphate; SHMT1, Serine hydroxymethyltransferase 1; SHMT2, Serine hydroxymethyltransferase 2; THF, tetrahydrofolate; AdoMet, S-adenosylmethionine; AdoHcy, 2S-adenosyl-L-homocysteine, Pi, phosphate.

activity of Umps. UMP is the precursor for all pyrimidine nucleotides. It is interesting to note that the fourth step links pyrimidine biosynthesis to the respiratory chain, making it a pacemaker for cell growth and division under limited oxygen conditions (Huang & Graves, 2003). Both purine and pyrimidine biosynthesis are associated with folate-mediated onecarbon metabolism (Figure 1). Folate is a water-soluble vitamin that occurs in different chemical forms distinguished by their oxidation state and specific types of one-carbon substitution, and plays an important role in methylation and DNA synthesis. The one-carbon metabolic cycle in the cytoplasm is required for the de novo synthesis of purines and thymidylate. In mammals, both the third and ninth steps of purine de novo biosynthesis use the cofactor N10-formylTHF as the source of the C(8) carbon atom, while the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine

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monophosphate (dTMP) is catalyzed by thymidylate synthase using N5,N10-methyleneTHF as a cofactor (Stover & Field, 2011). Regulation of de novo purine biosynthesis The purine de novo biosynthesis pathway is generally negatively regulated by several of its end products. Purine-5’-nucleotides, predominantly AMP and GMP, provide a negative feedback for the synthesis of PRPP by PRPP synthetase (Becker & Kim, 1987). Binding of A/GTP, A/GDP and A/GMP at allosteric sites inhibits the amidotransferase reaction catalyzed by PPAT. Purine biosynthesis is also regulated in the branch pathways from IMP to AMP or GMP (Hershfield & Seegmiller, 1976). The accumulation of excess ATP leads to accelerated synthesis of GMP, and excess GTP leads to accelerated synthesis of AMP. In addition, purine flux is co-regulated by coordinated repression at the transcriptional level of the genes that encode enzymes of the pathway as well as those in biosynthesis of substrates including glutamine, glycine and N10-formylTHF that are consumed in the pathway (Denis & Daignan-Fornier, 1998). Finally, proper nucleotide balance has to be maintained, probably via individual nucleotide sensing and adjustment mechanism at both the enzyme and gene level (Ljungdahl & Daignan-Fornier, 2012). As building blocks, nucleotides are required primarily during growth and division. Contrary to salvage enzymes, the purine biosynthesis enzymes display a variable activity during growth with peak values during periods of rapid cell division (Mayer et al., 1990). Furthermore, cells under quiescent conditions such as during nutrition starvation maintain only a low level of de novo biosynthesis through downregulation of most, if not all, purine biosynthesis genes at the transcriptional level (Ashrafi, Sinclair, Gordon, & Guarente, 1999). Yet, quiescent cells must act quickly upon re-entry into the cell cycle when nutrient availability is restored (Gray et al., 2004). This is only possible if pools of critical cellular components such as mRNAs are reserved (Brengues, Teixeira, & Parker, 2005; Meignin & Davis, 2010), and other cellular structures such as the actin cytoskeleton adapt to the stress when cells enter quiescence (Sagot, Pinson, Salin, & Daignan-Fornier, 2006). Stimulating cells (e.g., keratinocytes) with growth factors such as epidermal growth factor (EGF) can rapidly upregulate enzymes in the de novo pathways, resulting in accelerated nucleotide production to meet the demand for reentry into the cell cycle (Gassmann, Stanzel, & Werner, 1999).

Spatial organization of enzymes for metabolic flux and purinosome Inside mammalian cells the intracellular concentration of proteins can be as high as 200–300 mg/ml for mammalian cells, resulting in a high level of molecular crowding (Fulton, 1982; Luby-Phelps, 2000). However, some proteins are expressed at low numbers per cell and as such their movements are greatly restricted by the crowded surroundings (Nagaraj et al., 2011). Yet, cells are capable of multitasking and can accomplish their tissue specific functions as well as robust and specific responses. This is made possible by expressing cell-specific receptors and their interacting proteins (Lattin et al., 2007; Salazar, Chen, & Rockman, 2007), and by tight regulation of the temporal and spatial gradients of signaling molecules and networks in cells (Kholodenko, 2006; Zhou, Rivas, & Minton, 2008). Most cellular components are functionally interdependent via various interactions including molecular binding, biochemical

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reactions, expression regulation and multi-protein assemblies. Functional assemblies such as the T-cell receptor signalosome, the Wnt signalosome and the adhesion signalosome are examples of this level of protein organization and have been observed and proposed to play important roles in regulating cell signaling and functions (Lee, DeLoache, & Dueber, 2012). Substrate channeling has been proposed to be common to metabolic pathways including purine de novo biosynthesis. The channeling refers to the passing of the metabolic intermediate of one enzyme directly to another enzyme or active site. Substrate channeling is a rapid and efficient means to prevent the release of unstable intermediates, as well as to prevent an intermediate from being consumed by competing reactions catalyzed by other enzymes (Huang et al., 2001). Several mechanisms have been proposed to explain substrate channeling. These include restricted diffusion and the presence of metabolons. Physical barriers such as cell plasma membranes, intracellular membranes and the dense mesh of cytoskeletal networks can limit the diffusion of metabolic intermediates. However, a more efficient means is the formation of dynamic multienzyme complexes, termed metabolons (Srere, 1987). These metabolons can transiently and reversibly assemble and disassemble within the cytoplasm or cellular organelles. In the purine de novo biosynthetic pathway, there are two chemically unstable intermediates including 5-phosphoribosylamine (PRA) and formylglycinamidine ribonucleotide (FGAM), while both carboxyaminoimidazole ribonucleotide (CAIR) and aminoimidazole ribonucleotide (AIR) are putatively unstable (Schendel, Cheng, Otvos, Wehrli, & Stubbe, 1988). The presence of unstable intermediates and multifunctional enzymes is indicative of substrate channeling of purine de novo biosynthesis, thus the possibility to form multienzyme complexes (Srere, 1987). Early experiments indicate that enzymes in this pathway can be co-purified during isolation procedures in avian liver and human lymphocytes (McCairns, Fahey, Sauer, & Rowe, 1983). However, other contradictory data do exist; evidence provided suggests that it is unlikely that a stable interaction between PPAT and GARS is formed, between which unstable PRA has to channel (Rudolph & Stubbe, 1995). In addition, it seems that substrate channeling does not occur in the last two steps of the de novo pathway where substrate binding sites do not communicate (Bulock, Beardsley, & Anderson, 2002). However, these experiments were predominantly carried out with bacterial forms of the enzymes in vitro and may not be indicative of what may occur in vivo in mammalian systems. Given the macromolecular crowding that can promote the association of proteins (Zhou et al., 2008), it is still possible that enzymes in this pathway can assemble into functional complexes to facilitate substrate flux in living cells under specific conditions (Gooljarsingh, Ramcharan, Gilroy, & Benkovic, 2001). Recently, An et al. (2008) found that, when transiently expressed as fluorescent protein-tagged enzymes in HeLa cells, all six enzymes of the purine de novo biosynthesis pathway formed discrete clusters in the cytoplasm of living cells with high yield under purine-depleted conditions, but not in purine-rich media. Furthermore, both endogenous and green fluorescent protein (GFP) tagged TrifGART were co-localized into clusters with orange fluorescent protein (OFP)-tagged FGAMS when co-expressed in the purine-depleted media. In addition, they found that the fluorescent pattern of phosphoribosyl formylglycinamidine synthase-green fluorescent protein ((hFGAMSGFP) can undergo a dynamic and reversible transition from clusters to diffusive fluorescence when the purine-depleted media were exchanged with the purine-rich medium. Furthermore, the association and dissociation of purinosomes were found to be

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correlated with the rate of de novo purine biosynthesis in HeLa cells – an increased purine biosynthesis was observed when purinosomes were formed by monitoring 14C-glycine incorporation into the pool of cellular purines (An, Deng et al., 2010). Given the sensitivity and reversible dynamics to different purine levels, the metabolic multienzyme complex observed was termed the ‘purinosome’. Of note, the purinosomes should not be confused with other commonly occurring fluorescent punctates, which may simply be a result of protein aggregation due to overexpression. It is quite common to observe such punctates with variable size and shape occurring in transiently transfected cells when expression of fluorescent proteins is high. Conversely, the purinosome clusters tend to distribute broadly throughout the cytoplasm and have relatively small and uniform geometry (Figure 2). In addition, a key diagnostic for purinosomes is their ability to reversibly assemble and disassemble under a variety of different conditions. Purinosomes can form in a variety of cell backgrounds under specific conditions. Besides HeLa cells, the formation of purinosomes has been observed in human liver carcinoma cell line HepG2C3A (An, Kyoung et al., 2010), human hepatocellular liver carcinoma cell line HepG2, sarcoma osteogenic cell line Saos-2, human embryonic kidney cell line HEK293, human skin fibroblasts and primary human keratinocytes cultured in purine-depleted media (Baresova et al., 2012). Interestingly, in human skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency, various mutations of ATIC and ADSL were found to destabilize purinosome to various degrees, and the ability to form these assemblies was found to correlate with clinical phenotypes of indi-

Figure 2. Confocal images of live HeLa cells cultured in purine rich medium before (a) and 4 h after (b) the treatment with the α2A-adrenergic receptor agonist oxymetazoline (100 nM). Note: The cells were transiently expressing hFGAMS-GFP as a purinosome marker. Images courtesy of Dr Songon An at University of Maryland Baltimore County.

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vidual ADSL patients (Baresova et al., 2012; Marie et al., 2004; Zikanova, Skopova, Hnizda, Krijt, & Kmoch, 2010). The purinosome complex contains not only enzymes in purine de novo biosynthesis, but also other enzymes and scaffold proteins. Recently, methenyltetrahydrofolate synthetase (MTHFS) was found to be co-localized with OFP-tagged hFGAMS or hTrifGART in HeLa cells in a cell-cycle-dependent manner (Field et al., 2011). This study also showed that MTHFS was modified by the small ubiquitin-like modifier (SUMO) protein, and mutation of the consensus SUMO modification sites on MTHFS eliminated the purinosome clusters under purine-deficient conditions. This is interesting from several aspects. First, while C1-THF synthase is the enzyme responsible for the synthesis of N10-formylTHF, MTHFS was found to be part of the purinosome. N10-formylTHF is a folate cofactor required for both TrifGART and ATIC, two enzymes in the purine de novo pathway (Stover & Field, 2011). MTHFS regulates folate turnover and accumulation, as increased MTHFS activity accelerates folate turnover rates and depletes cellular folate concentration (Anguera et al., 2003). As a storage form of THF cofactors, N5-formylTHF is mobilized back into the THF cofactor pool by MTHFS, which catalyzes the irreversible and ATP-dependent conversion N5-formylTHF to N5,N10methenylTHF. Thus, MTHFS seems to enhance purine biosynthesis by delivering N10-formylTHF to the purinosome in a SUMO-dependent fashion. Second, MTHFS expression is often elevated in animal tumors, leading to increased de novo purine synthesis, partial resistance to antifolate purine synthesis inhibitors and increased rates of folate catabolism in mammalian cells. Finally, folate-mediated one-carbon metabolism itself is well known to be compartmentalized in the mitochondria, nucleus and cytoplasm of eukaryotic cells (Tibbetts & Appling, 2010). The assembly and disassembly of multi-protein complexes may represent a general mechanism in various metabolic networks. Using phenotypic screening in conjunction with immunofluorescence and mass spectrometry, Narayanaswamy et al. (2009) found that many proteins formed punctate cytoplasmic foci when expressed in yeasts grown to stationary phase. These proteins are generally involved in intermediary metabolism and stress response. In particular, the purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. It is possible, however, that these yeast phenotypes may represent functionally inactive aggregates with respect to metabolic flux. Recently, Noree, Sato, Broyer, and Wilhelm (2010) found that in yeast glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex all can be recruited to microfilaments, forming long enzyme-decorated strings. In particular, recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity. CTP synthase makes CTP, a building block for DNA and RNA. In addition, CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. These long enzyme filaments may provide a unique cellular mechanism to turn enzymes on and off en masse (Kwok, 2011). In addition, Anderson, Woeller, Chiang, Shane, and Stover (2012) also found that thymidylate de novo biosynthesis occurs in the nucleus, and a multienzyme complex consisting of serine hydroxymethyltransferase 1 and 2 (SHMT1 and SHMT2), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) is formed and associated with the nuclear lamina and the DNA replication machinery.

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Regulation of purinosome dynamics The spatial distribution and metabolic activity of purinosomes in the cytoplasm of HeLa cells seems to be physically controlled by a microtubule network, as disruption of microtubules, but not actin filaments, caused dispersion of purinosomes and led to an attenuated metabolic flux of de novo purine biosynthesis under purine-depleted conditions (An, Deng et al., 2010). This is interesting when compared to the multienzyme complexes associated with glycolysis. The glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase and pyruvate kinase were found to organize into multi-enzyme complexes on the inner surface of the human erythrocyte membrane (Campanella, Chu, & Low, 2005). Most glycolytic enzymes can reversibly bind to both actin filaments and microtubules (Cassimeris et al., 2012). However, glycolysis-driven ATP production was found to be specifically associated with actin cytoskeleton dynamics (Bereiter-Hahn, Stübig, & Heymann, 1995). By contrast, microtubules can regulate multiple other steps in glycolysis, including insulin-dependent transport of glucose transporter 4 to the plasma membrane, translation of the mRNA encoding hypoxia inducible factor-1a, and transport of metabolites into and out of mitochondria through the binding of tubulin dimers to the voltage-dependent anion channel of the mitochondrial outer membrane (Cassimeris et al., 2012). The dynamics of purinosomes also seem to be regulated by cell signaling. Cell cycle and proliferation are tightly regulated processes controlled by many kinase cascades. Given the important role of purine biosynthesis in DNA replication, it is reasonable to believe that the de novo pathway, and thus purinosomes, is subject to kinase regulation. This is supported by several lines of evidence. First, recent proteomic studies suggest that hTrifGART and hFGAMS are likely to be phosphorylated at binding motifs recognized by 14-3-3 proteins that play important roles in many biological processes, including cell-cycle regulation and signal transduction (Campanella et al., 2005). Second, treating keratinocytes with either keratinocyte or EGF led to upregulation of all the enzymes in the de novo pathway, wherein both growth factors are known to activate the PI3K/Akt pathway (Gassmann et al., 1999). Third, overexpression of myristoylated, constitutively active Akt in Rat1a fibroblasts led to a three-fold increase in intracellular ATP concentration (Hahn-Windgassen et al., 2005), suggesting that constitutively active Akt may influence purine de novo biosynthesis. Recently, the PI3K/Akt cascade was indeed found to regulate directly the synthesis of purine nucleotides via both the de novo and salvage pathways (Wang et al., 2009). Akt appears to regulate de novo purine synthesis by regulating PRPP availability and ATIC activity, while inhibiting Akt activity suppresses HPRT-mediated salvage activity. Fourth, several small molecule inhibitors of casein kinase II (CK2) including 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), tetrabromocinammic acid (TBCA) and ellagic acid were found greatly to promote purinosomes in HeLa cells under purine-rich conditions via a CK2-mediated mechanism as confirmed using RNA interference (An, Kyoung et al., 2010; Verrier et al., 2011). CK2 is a serine/threonineprotein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. In an interesting contrast, another known CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), was found to cause the dissociation of DMAT-promoted or purine-depletioninduced purinosome clusters in HeLa cells, suggesting an effect via a non-CK2 mediated pathway. This was further confirmed using label-free dynamic mass redistribution

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(DMR) whole cell assays (Verrier et al., 2011). In the confluent native HeLa cells DMAT was found to trigger a positive DMR that is sensitive to knockdown of CK2 by RNAi, while TBB caused a negative DMR that is mostly insensitive to CK2 knockdown. Furthermore, cross-sensitization of DMR responses between DMAT and TBB indicate that both kinase inhibitors oppositely regulate the same cellular process (i.e., the purinosome). Of note, many, if not all, kinase inhibitors are known to have polypharmacology (Pagano et al., 2008; Deng, Hu, & Fang, 2011; Fang, 2012). Several CK2 inhibitors also display activity beyond kinases; for instance, ellagic acid and TBCA were recently found to be agonists for GPR35, an orphan GPCR (Deng, Hu, Ling, Ferrie, & Fang, 2012; Deng & Fang, 2012). Nonetheless, these studies suggest that the purinosome dynamics may be under the control of cell signaling. G-protein-coupled receptors and mitogenic signaling G-protein-coupled receptors (GPCRs) represent the largest class of proven drug targets (Imming et al., 2006). It has been estimated that  27% of the 1357 unique drugs in the market before 2006 are GPCR ligands that are known to interact with at least 80 unique members of this receptor family (Swinney & Anthony, 2011; Rask-Andersen, Almen, & Schioth, 2011); more than a quarter of the 100 top-selling drugs are GPCR ligands (Zambrowicz & Sands, 2003). GPCRs also represent the largest family of druggable targets in the human genome, with  360 non-chemosensory receptors that are predicted to bind endogenous ligands (Chung, Funakoshi, & Civelli, 2008; Overington, Al-Lazikani, & Hopkins, 2006; Russ & Lampel, 2005). In light of their past success, their large number, widespread distribution and important roles in physiology GPCRs have been and continue to be the frequent targets of drug discovery campaigns (Conn, Ulloa-Aguirre, Ito, & Janovick, 2007; Thompson, Burnham, & Cole, 2005). All GPCRs consist of seven α-helical transmembrane-spanning domains joined by intra- and extracellular loops (Palczewski et al., 2000). GPCRs are expressed in virtually all tissues, but with distinct expression patterns (Salazar et al., 2007). Effectors for GPCRs are extremely diverse, ranging from light to ions, biogenic amines, amino acids, bioactive lipids, growth factors, hormones, metabolites, odorant molecules, peptides and proteins (Ji, Grossman, & Ji, 1998), permitting GPCRs to control a wide variety of physiological processes, such as cell proliferation, chemotaxis, inflammation, metabolism and neurotransmission (Conn et al., 2007). In the past two decades, advances in genomics and molecular genetics have further solidified the concept that GPCRs are implicated in almost every major disease class, including asthma, cancer, cardiovascular diseases and inflammation (Rohrer & Kobilka, 1998; Schöneberg, Schulz, & Gudermann, 2002; Dorsam & Gutkind, 2007; Lappano & Maggiolini, 2011). GPCRs participate in a multitude of cell-signaling pathways, primarily mediated through a subset of the 16 heterotrimeric G-protein subtypes (Neves, Ram, & Iyengar, 2002; Saulière et al., 2012). These G proteins are functionally grouped into four broad classes including Gs, Gi, Gq, and G12 (Figure 3). The consensus model concerning GPCR signaling assumes that agonist binding to the monomeric form of a receptor switches the receptor from an inactive state to an active state, thus promoting signaling through the activation of an associated heterotrimeric G protein (Oldham & Hamm, 2008). In this simple model, receptor signaling is described as a linear cascade starting from activation of the receptor to subsequent activation of G protein and enzymes at the plasma membrane. This leads to alterations in the levels of intracellular second messengers such as cAMP and activation of kinase cascades such as protein kinase A

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Amino acid, metabolite, lipid, nucleotide, biogenic amine, peptide, protein

G12 Gi

Gq

Gs

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IMP production Purinosome

Nucleus

Figure 3. The diversity of G protein-coupled receptor (GPCR) ligands and signaling pathways. Note: All four major classes of G-protein-mediated signaling pathways have been linked to the regulation of gene expression through transmission of mitogenic signals directly to the nucleus. In HeLa cells only Gi-mediated signaling via several endogenous GPCRs promotes purinosome formation. IMP, inosine 5’-monophosphate.

(PKA), which ultimately governs multiple cellular machines including ion channels, transcription factors, cytoskeletal proteins and metabolic enzymes. Mounting evidence accumulated in the past decades indicates that GPCR signaling is far more complicated than initially thought. At present, GPCRs are well known for their ability to mediate pleiotropic signaling through both G-protein-dependent and -independent mechanisms. The promiscuity of G proteins makes it possible for a single GPCR to couple to and signal via multiple G proteins (Hermans, 2003). A single GPCR can also perform isotype-specific functions through unique interaction partners and mediate G-proteinindependent signaling cascades such as β-arrestin-mediated signaling (DeWire, Ahn, Lefkowitz, & Shenoy, 2007; Ritter & Hall, 2009; Rajagopal, Rajagopal & Lefkowitz, 2010). In addition, many receptors can form signaling complexes via oligomerization (Bouvier, 2001) and interactions with other classes of proteins (Bockaert, Fagni, Dumuis, & Marin, 2004). Many GPCR agonists act as potent mitogens to promote normal and abnormal cell proliferation in addition to their role as transducers engaged in tissue-specific and post-mitotic functions in differentiated cells. GPCR agonists such as angiotensin II, bombesin, bradykinin, lysophosphatidic acid (LPA), thrombin and vasopressin are tumorigenic and can promote cellular transformation (reviewed in Rozengurt, 2007). The classical model concerning GPCR mitogenic signaling describes that agonist binding activates multiple receptor signal transduction pathways that ultimately lead to cell proliferation through transmission of the mitogenic signal directly to the nucleus (Rozengurt, 2007). GPCR mitogenic signaling is known to be mediated through all four G-protein families as well as through β-arrestins in a cell context-dependent manner. This accounts for their proliferative, anti-apoptotic and promigratory effects. Many growth-promoting GPCR agonists can activate a large number of kinases, such as protein kinase D (Rozengurt, Rey, & Waldron, 2005; Sinnett-Smith, Zhukova, Hsieh, Jiang, & Rozengurt, 2004), focal adhesion kinase (FAK; Jiang, Sinnett-Smith, &

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Rozengurt, 2007), the target of rapamycin (mTOR; Wullschleger, Loewith, & Hall, 2006), EGF receptor (Prenzel et al., 1999) and mitogen-activated protein kinase (MAPK; Lappano & Maggiolini, 2011). Some of these kinase cascades may act in a synergistic and combinatorial fashion to promote normal cell growth and cancers. G-protein-coupled receptors and the purinosome As mentioned previously, GPCR mitogenic signaling is thought to propagate through several kinase cascades to regulate directly the nuclear expression of growth-promoting genes (Dorsam & Gutkind, 2007). To discover the upstream receptor signaling pathways that regulate purinosome dynamics, we recently developed a methodology combining a label-free whole-cell phenotypic assay with immunofluorescence imaging and molecular biology techniques (Verrier et al., 2011). The label-free whole-cell phenotypic assay is enabled by microplate-based resonant waveguide grating (RWG) biosensors (Fang, 2007). The biosensor is a label-free optical biosensor that acts as a highly sensitive filter to select the light at a specific wavelength and couple it to the biosensor waveguide thin film, so an electromagnetic field can be generated and extended towards the cells cultured on the waveguide surface (Fang, Ferrie, Fontaine, Mauro, & Balakrishnan, 2006). The light with the maximal coupling efficiency is termed the resonant wavelength, which is a direct function of local refractive index at the sensor–cell interface. The local refractive index is directly proportional to the cellular mass density. Therefore, the biosensor can detect ligand-induced DMR within the cell layer by monitoring the shift of the resonant wavelength of the biosensor in real time. The DMR arising from a ligand-receptor interaction is an integrated whole-cell response that reflects receptor signaling pathway and cell decision-making processes (Fang, 2011, 2010, 2011). Compared with conventional molecular assays (Galandrin, Oligny-Longpre, & Bouvier, 2007), DMR assays are advantageous in consideration of their ability to provide a holistic view of receptor signaling in real time at the wholecell level with non-invasive and high-throughput measurements (Rocheville & Jerman, 2009; Kenakin, 2010; Fang, 2011). Using this methodology we discovered the regulative role of several endogenous Gi-coupled receptors, such as α2A-adrenergic receptor (α2A-AR) and purinergic receptors, in purinosome dynamics (Verrier et al., 2011). Receptor panning using known GPCR agonists identified a number of GPCRs that are endogenously expressed and functional in HeLa cells. Screening GPCR agonists revealed that a subset of agonists suppressed the DMAT response (stimulation of purinosome formation), but potentiated the TBB responses (disruption of purinosomes). Pharmacological profiling, together with immunofluorescence, showed that it is α2A-AR, but not β2-AR, whose activation promotes purinosome in purine-rich medium (Figure 2). Pathway deconvolution using chemical biology and molecular genetics techniques also showed that it is the Gi pathway, but not Gs pathway, that is involved in purinosome formation. The ability to promote purinosome formation was found to be a general mechanism for several Gi-coupled receptor agonists, including purinergic P2Y receptor agonists ATP, ADP and UDP, an LPA receptor agonist LPA, and prostaglandin receptor agonists PGE2 and PGD2. The cognate receptors for these agonists were indeed expressed and functional in HeLa cells, as confirmed using real-time PCR and DMR receptor-panning studies. Together, these results point to a GPCR-controlled cellular mechanism to regulate purinosome assembly. This linkage is an alternative route for GPCR mitogenic signaling (Mohr & Kostenis, 2011).

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Implications for drug discovery Nucleotide de novo biosynthesis is often upregulated in cancer cells to provide building blocks for the synthesis of RNA, DNA and other effectors (Denkert et al., 2008). Blocking nucleotide metabolism has been quite a successful strategy for the development of anticancer, anti-inflammatory, antiviral and antimicrobial drugs (Parker, 2009). Given the importance of nucleotides in DNA replication and RNA synthesis, it is difficult for the cell to replicate the genome in the presence of a complete block of nucleotide de novo synthesis. Since most adult cells are quiescent and the quiescent cells at the resting state generally do not perform extensive DNA duplication, inhibiting nucleotide metabolism affords some level of selectivity, thus offering a therapeutic window to develop effective anticancer drugs. To date there are 15 FDA-approved drugs that are purine and pyrimidine antimetabolites, representing over 20% of all approved oncology drugs (Wishart et al., 2006). Given the more toxic side effects associated with blockage of the pyrimidine pathway, the purine pathway is the favored target for inhibition. Developing drugs based on nucleoside analogues is still a productive area for discovering new drugs for the treatment of cancer and other diseases (Parker, 2009). On the other hand, GPCRs have been implicated in cancer (Dorsam and Gutkind, 2007; Lappano & Maggiolini, 2011). A recent study showed in addition that in mice the activation of b2 -AR by catecholamines mediates both Gs-PKA and β-arrestin signaling pathways to trigger DNA damage and suppress p53 levels, both of which, in turn, synergistically result in the accumulation of DNA damage (Hara et al., 2011). This study provides a possible cellular mechanism for cancer development. However, there is no single GPCR drug that has been approved for treating cancer and other malignant diseases. The GPCR-regulated purinosome may represent a new entry point to develop anticancer GPCR therapeutics. Abnormal catabolism of the purines also leads to other types of diseases ranging from mild to serve disorders. Clinical manifestations of abnormal purine catabolism arise from the insolubility of the degradation byproduct, uric acid. Both hyperuricemia and gout are the result of excess purine production and deficiencies in the consequent catabolism or to a partial deficiency in the salvage enzyme, HGPRT. Hyperuricemia is a condition associated with excess uric acid in blood plasma. Gout is a kind of arthritis that occurs when uric acid builds up in blood and causes joint inflammation and arthritis mostly due to the crystals engaging the caspase-1-activating inflammasome (Terkeltaub, 2010). Allopurinol, a structural analog of hypoxanthine and a potent xanthine oxidase inhibitor, is effective for treating most forms of gout. Although it is unknown whether GPCRs play a role in gout, high uric acid in the blood stream can result from insulin resistance. A recent study showed that some GPCRs can cross-talk with insulin/ insulin-like growth factor-1 pathways in pancreatic cancer cells, leading to enhanced signaling, DNA synthesis and proliferation in a mTOR complex 1-dependent mechanism (Rozengurt, Sinnett-Smith, & Kisfalvi, 2010). In addition, overexpression of α2AAR in a rat model was found to mediate adrenergic suppression of insulin secretion, and to increase type 2 diabetes risk (Rosengren et al., 2010). Pharmacological receptor antagonism, silencing of receptor expression or blockade of downstream effectors all rescued insulin secretion in congenic islets. These results indicate that the GPCR regulation of the purinosome, in addition to a potential anticancer effect, may present a possible mechanism to develop new therapeutics for treating inflammatory diseases such as gout, arthritis and diabetes.

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Conclusions Nucleotide metabolism is essential to cell function. Enzymes in in the purine de novo biosynthetic pathway and some related pathways can self-assemble into multi-enzyme complexes in living cells under specific conditions. The purinosome may be essential to purine de novo biosynthesis and its related biological processes. Early studies suggest that the purinosome is associated with accelerated purine synthesis, and is under regulation of cell signaling including GPCR signaling. The GPCR-controlled purinosome formation may represent a druggable mechanism to develop novel therapeutics. However, further elucidation of the composition, molecular organization, function(s) and regulation processes of purinosomes is essential to validate such a mechanism.

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G-protein-coupled receptor regulation of de novo purine biosynthesis: a novel druggable mechanism.

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of ce...
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