Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function. Over the past half-century, studies have highlighted the importance of dynamic remodelling of enzyme quaternary structure in the form of filamentous structures found in mammalian, yeast, and bacterial cells (see [1-5] and references therein). Delineation of the function of these intracellular assemblies of metabolic enzymes [often appearing as filaments or bundles of filaments, which we will refer to as ‘filaments’ or ‘assemblies’, but are sometimes referred to as ‘rod and rings’ (RR) [6] or ‘cytoophidia’ [7]] has only just started, especially with respect to their role in enzyme regulation [8]. CTP synthase (CTPS) [7, 9, 10] and inosine-5′-monophosphate (IMP) dehydrogenase (IMPDH) [11] are examples of such enzymes that undergo filament formation both in vitro and in vivo. Most intriguingly, the filaments of both enzymes co-assemble in mammalian cells [6]. CTPS catalyses the rate-limiting step for the de novo formation of CTP, that is, the ATP-dependent amination of UTP using either l-glutamine or free ammonia as the source of nitrogen (Fig. 1) [12]. Two isoforms, CTPS1 and CTPS2, have been identified, which share 74% amino acid identity and function as tetramers. IMPDH catalyses the (NAD+)-dependent oxidation of IMP to xanthosine-5′-monophosphate, which is the rate-limiting step in the de novo biosynthesis of guanine nucleotides [13]. Again, two isoforms, IMPDH1 and IMPDH2, have been identified. They share 84% amino acid sequence identity and function as octamers. Because rapidly proliferating cells require CTP and GTP, both allosterically controlled enzymes are targets for the development of anticancer, antiviral, and immunosuppressive chemotherapies. In this issue of The FEBS Journal, Liu, Sung, and co-workers [14] explore the morphology and dynamics of CTPS1/IMPDH2-containing assemblies in live human cells. Recent examination of the morphology and subcellular localization of CTPS1/IMPDH2 assemblies revealed rods of 5–10 μm and ring structures of 2–5 μm in diameter, and no obvious preference for any specific cellular compartment [8, 15]. Rods found in the nucleus tend to be small, and larger assemblies of rods and rings can be found distributed throughout the cytoplasm [8]. Factors governing the assembly of these structures are unclear, but fluctuations in intracellular NTP levels appear to be involved. Human CTPS filaments form bound to substrates (UTP and ATP) and disassemble with the abundance of the product (CTP) [16]. Similarly, human IMPDH2 filaments can form bound to activators (IMP and NAD+) [17] and are dismantled by GTP in vivo [18]. CTPS1/IMPDH2 assemblies are induced by inhibitors of these enzymes, but whether the assembly follows inhibitor binding or depletion of CTP and GTP pools has not been fully explored [6, 16-20]. Treatment with either of the IMPDH inhibitors ribavirin or mycophenolic acid (MPA) produces primarily IMPDH2-based assemblies, whereas treatment with 6-diazo-5-oxo-l-norleucine (DON) alone, an inhibitor of purine and pyrimidine biosynthesis (Fig. 1), furnishes a mixture of CTPS-based, IMPDH2-based, and hybrid assemblies. Both enzymes may conform to ‘active’ configurations within these assemblies [21]. In this issue, Chang et al. [14] describe a HeLa cell line expressing both OFP-IMPDH2 and CTPS1-eGFP and analyse the assembly of these fusion proteins in real time using fluorescence microscopy. Capturing elegant images and videos, the authors demonstrate the dynamic nature of rod- and ring-like assemblies of IMPDH2 and CTPS1 in vivo when human cells are subjected to inhibitors of these enzymes (Fig. 2). The observed fluidity of the structures suggests that the differently shaped assemblies are essentially interchangeable. Although these assemblies often moved in synchrony, consistent with their being associated, they also exhibited the ability to spontaneously separate, suggesting that the association is loose. Treatment of the transfected HeLa cells with ribavirin or MPA rapidly induced IMPDH2 assembly, but failed to stimulate CTPS1 polymerization. Thus, both IMPDH2 inhibitors likely have no effect on the determinants of CTPS1 filament formation and/or IMPDH2 assemblies are otherwise incapable of nucleating CTPS1 assembly on their own. Interestingly, upon treatment with the CTPS inhibitor, DON, the authors observed rapid formation of hybrid CTPS1/IMPDH2-containing assemblies. Although DON depletes CTP pools by irreversible inhibition of CTPS, IMPDH2 assembly may have been induced by depletion of guanosine nucleotide pools following similar inhibition of the amidotransferases of purine biosynthesis [22, 23]. Supplementation with exogenous guanosine disassembled the IMPDH2 filaments, but left CTPS1 filaments untouched (Fig. 2). Treatment of cells with the CTPS1 inhibitor, 3-deazauridine (DAU; metabolized to the corresponding 5′-triphosphate), initiated sequential CTPS1/IMPDH2 assembly. First, CTPS1 filaments were observed followed by IMPDH2 assemblies 30 min post-treatment (Fig. 2); however, DAU does not inhibit purine biosynthesis and IMPDH2 structures later disassembled, presumably once guanosine nucleotide pools were restored [19]. That inducers of CTPS1 assemblies caused IMPDH2 polymerization, but not the converse, is evidence that CTPS1 may stimulate IMPDH2 assembly. Elucidation of the mechanism might be aided by DAU treatment of cells expressing OFP-IMPDH2 and a CTPS1 variant that cannot polymerize, like that used by Lynch et al. [16]. Overall, it appears that CTPS1 and IMPDH2 filaments behave as separate and distinct entities with no mixing of the proteins being observed within the individual filaments and neither is required for the assembly of the other [4, 19]. Fluorescence recovery after photobleaching of the mixed assemblies revealed uniform recovery of fluorescence suggesting that the assemblies may continuously renew subunits and that there was no polarity to the structures. Finally, using stimulated emission depletion super-resolution microscopy, the authors demonstrate that the CTPS1 and IMPDH2 filaments are separate but aligned or intertwined with one another in the mixed assemblies. Interestingly, gaps were observed between aligned filaments, again consistent with the notion that the CTPS1 and IMPDH2 filaments associate loosely or require involvement of a third party such as a membrane, or another protein, to act as the ‘glue’ within the assembly [6, 24]. The involvement of cytoskeletal elements is ambiguous since assemblies in human cells composed of CTPS1 and IMPDH2 filaments were not enriched in actin, tubulin, or vimentin [6, 10], nor did they co-localize with Golgi complexes or centromeres, but Liu et al. [7] determined that CTPS filaments were associated with the microtubular network and Golgi in Drosophilia cells. At present, the role of most higher-order assemblies of metabolic enzymes in cells remains unclear. While quaternary structures facilitate the channelling of substrates and reactive intermediates between individual subunits, and enzyme complexes may improve metabolic efficiency via direct transfer of metabolites, as with the purinosome [25], these are likely not the roles of CTPS1/IMPDH2 assemblies. More likely roles of CTPS and IMPDH filaments, either individually or in a mixed-assembly, include regulation by cooperative interactions and allosteric effectors, altering the conformation to affect post-translational modification (e.g., phosphorylation [4, 26], methylation [27], and/or ubiquinylation [28]), acting as a platform for recruitment of other enzymes and regulatory proteins, serving as protein depots that can act to rapidly supply active enzyme when required, and/or serving as a mechanism to sequester the enzymes either inside or outside the nucleus [3, 4]. CTPS is regulated by the positive allosteric effector GTP [29] (Fig. 1), thereby affording a mechanism by which elevated levels of GTP can effect an increase in the CTP pool. However, a direct reciprocal regulation of the purine and pyrimidine nucleotide biosynthetic pathways has not been observed, except through sharing the pool of phosphoribosyl pyrophosphate [19], wherein elevated UDP/UTP levels activate de novo purine synthesis via UDP/UTP-dependent inhibition of carbamoyl phosphate synthase (Fig. 1). It is tempting to speculate that the interaction between IMPDH2 and CTPS1 in filamentous assemblies could provide such a regulatory mechanism, especially considering that both enzymes catalyse the rate-dependent steps in their biosynthetic pathways, that both enzymes are present together in intracellular assemblies, and that nucleotide biosynthesis is tightly regulated by the cell [6]. However, the apparent loose association and gaps between assemblies disfavour this hypothesis, unless a ‘bridging’ protein is present. Currently, cryo-EM structures are available for human CTPS1 filaments at 6.1-Å resolution [16] and human IMPDH2 filaments at 8.7–20-Å resolution [17]. Filaments of eukaryotic CTPS1 are active [16, 30] and are composed of stacked tetramers that are rotated slightly with respect to each other, generating a helical filament [16]. IMPDH2 filaments, formed from stacked octamers, assume four helical filament types with different conformational states of the octamers: expanded (promoted by substrate binding), collapsed (induced by binding of GTP), bent, and ‘poorly aligning’. The gaps observed by Chang et al. in the present study could arise from the bent conformation of IMPDH2 or a third binding determinant that has not yet been identified. Since IMPDH2 filaments are catalytically active and capable of allosteric regulation through GTP binding [17], the collapsed conformational state of IMPDH2 could serve as a physical signal to modulate the activity of CTPS1. Similarly, the conformational change effected by GTP binding to CTPS1 could act as a physical signal to modulate the activity of IMPDH2. Considering the helical nature of CTPS1 and IMPDH2 filaments, a side-by-side arrangement of either filaments or fibres may not yield a uniform set of binding interactions. Indeed, the loose nature of the mixed assemblies observed by Liu and co-workers would be consistent with a lack of specific and cooperative binding interactions. Certainly, it will be interesting to obtain cryo-EM structures of mixed CTPS1/IMPDH2 assemblies, and potential ‘bridging’ proteins, to learn more about the interactions between filaments at near-atomic levels. The studies reported by Chang et al. [14] clearly illustrate the dynamic characteristics of CTPS1/IMPDH2-containing filaments in live cells, as well as the independent behaviour of the filaments derived from CTPS1 and IMPDH2. Establishment of the HeLa cell line bearing fluorescently-tagged CTPS1 and IMPDH2 furnishes a useful tool for future investigations at higher resolution to further probe the effect of varying treatments (e.g., other chemotherapy drugs or altered levels of nutrients), to explore the relationship between assembly morphologies and function, and to develop inhibitors and promoters of the assembly process. Delineating the physiological role of intracellular, higher-order assemblies of metabolic enzymes is a rich area for investigation.

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