A New View into the Regulation of Purine Metabolism: The Purinosome

Abstract

Other than serving as building blocks for DNA and RNA, purine metabolites provide a cell with the necessary energy and cofactors to promote cell survival and proliferation. A renewed interest in how purine metabolism may fuel cancer progression has uncovered a new perspective into how a cell regulates purine need. Under cellular conditions of high purine demand, the de novo purine biosynthetic enzymes cluster near mitochondria and microtubules to form dynamic multienzyme complexes referred to as 'purinosomes'. In this review, we highlight the purinosome as a novel level of metabolic organization of enzymes in cells, its consequences for regulation of purine metabolism, and the extent that purine metabolism is being targeted for the treatment of cancers.

Keywords: metabolon; purine metabolism; purinosome.

Figure 1. Purine metabolic pathways and their crosstalk with other metabolic processes

The de novo purine biosynthetic pathway in humans consists of 10 highly conserved steps (green) that transforms phosphoribosylpyrophosphate (PRPP), generated through the pentose phosphate pathway (blue), into inosine 5′-monophosphate (IMP). Six enzymes catalyze these ten steps and include PRPP amidotransferase (PPAT, EC 2.4.2.14), trifunctional phosphoribosylglycinamide synthetase (GARS, EC 6.3.4.13)/phosphoribosylglycinamide formyltransferase (GAR Tfase, EC 2.1.2.2)/ phosphoribosylaminoimidazole synthetase (AIRS, EC 6.3.3.1) (GART), phosphoribosyl formylglycinamidine synthase (FGAMS, EC 6.3.5.3), bifunctional phosphoribosyl aminoimidazole carboxylase (CAIRS, EC 4.1.1.21)/phosphoribosyl aminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS), adenylosuccinate lyase (ADSL, EC 4.3.2.2), and bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICAR Tfase, EC 2.1.2.3)/IMP cyclohydrolase (IMPCH, EC 3.5.4.10) (ATIC). Pathway cofactor, 10-formyltetrahydrofolate (10-fTHF) is a product of one-carbon metabolism (denoted with a *, for biosynthetic pathway see Figure 3). Intermediate SAICAR was shown to allosterically activate pyruvate kinase isoform M2 (PKM2, EC 2.7.1.40) in glycolysis (red) [26-28]. AICAR is also a byproduct of histidine biosynthesis (purple). Downstream purine biosynthesis of IMP requires the use of IMP dehydrogenase (IMPDH, EC 1.1.1.205), GMP synthase (GMPS, EC 6.3.5.2) to make GMP whereas AMP can be generated by reactions catalyzed by adenylosuccinate synthase (ADSS, EC 6.3.4.4) and ADSL. Purine salvage (orange) can also be used to generate IMP and GMP using hypoxanthine-guanine phosphoribosyl transferase (HPRT, EC 2.4.2.8) and AMP from adenine phosphoribosyl transferase (APRT, EC 2.4.2.7).

Figure 2. Purinosome assembly in cells is proposed as a stepwise process

While the exact triggers for purinosome formation are not known, diffusion and protein-protein interaction studies have provided insight into how purinosomes may assemble in cells under purine-depleted growth conditions. (B) The first three enzymes in the de novo purine biosynthetic pathway (PPAT, GART, and FGAMS) form the core of the purinosome and assemble first before secondary complexes of [PAICS·ADSL] and [ATIC] interact with the core. (C) Several proteomic studies have uncovered interactions between the enzymes in the de novo purine biosynthetic pathway [33, 39-43]. Captured here is the oligomeric state of the active form of each enzyme in the de novo purine biosynthetic pathway and those protein-protein interactions reported between them where the interaction strength (edge thickness) corresponds to the number of studies referencing the interaction. Downstream enzymes of IMP, IMPDH and ADSS, were also shown to be part of the purinosome; however, no protein-protein interactions between these enzymes and the de novo purine biosynthetic enzymes have been reported [36]. Likewise, Hsp90 and CK2 have been demonstrated to influence purinosome formation in cells by unknown mechanisms [38, 48].

Figure 3. mTOR is involved in purinosome localization near mitochondria likely through ATF-directed MTHFD2 expression

(A) Using FGAMS-mEos2 as a purinosome marker, the colocalization of purinosomes (red) with mitochondria (green) was determined in HeLa cells under purine-depleted conditions using Stochastic Optical Reconstruction Microscopy (STORM). Scale bar = 5 μM. (B) Those purinosomes deemed to be colocalized with mitochondria shown in magenta. Details of how colocalization was determined can be found in French, J.B. et al. [61]. Images courtesy of Sara A. Jones and Xiaowei Zhuang. (C) Our working hypothesis on how mTOR likely controls purinosome-mitochondria colocalization. mTOR regulates expression of MTHFD2, which encodes the mitochondrial dehydrogenase (MTHFD2) responsible for formate release into the cytoplasm [64]. Formate is readily converted into 10-formyltetrahydrofolate (10-fTHF) by the enzyme MTHFD1 where it is used as a cofactor for GAR and AICAR Tfase reactions within the purinosome. Inhibition of mTOR with rapamycin, causing a decrease in purinosome-mitochondria colocalization, results in a decrease in MTHFD2 expression and formate release suggesting that one reason for the observed colocalization is to compartmentalize the enzymes near areas of high formate concentration [89].