Glutamine deprivation initiates reversible assembly of mammalian rods and rings

Abstract

Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (<2 μm) assembled after 24 h, and longer rods (>5 μm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

‘Rod and ring’ formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway

“…An emerging theme in RR research is that drugs that throttle GMP biosynthesis, either by directly inhibiting IMPDH (with mycophenolic acid or ribavirin) or indirectly by limiting essential precursor availability (by inhibiting glutamine synthetase with DON or methionine sulfoximine) likewise promote RR assembly (Calise et al, 2014b;Carcamo et al, 2011;Gou et al, 2014;Gunter et al, 2008;Ji et al, 2006). We demonstrated here that RR formation also occurs when cells are treated with methotrexate or aminopterin, which target dihydrofolate reductase.…”

“…Purine and pyrimidine nucleotides are mainly derived from amino acids, and we have demonstrated that depriving cells of glutamine leads to RR formation (Calise et al, 2014b). Moreover, irreversible inhibition of glutamine synthetase by methionine sulfoximine induces RR assembly.…”

“…In a previous study, we have shown that RRs induced by glutamine deprivation subsequently underwent extensive disassembly (often in 15 min) when cells were supplemented with either glutamine or guanosine (Calise et al, 2014b). When cells were deprived of serine (by maintaining them in MEM for 72 h) and then supplemented with either 0.4 mM serine or 1 mM guanosine for 15 min, 1 h or 4 h, we found a significant reduction in the percentage of cells containing RR compared to controls ( Fig.…”

“…His discovery that inosine 5′-monophosphate dehydrogenase (IMPDH, for which there are two variants, IMPDH1 and IMPDH2, in mammals), which catalyzes the reaction IMP+H 2 O+NAD + ⇌XMP+NADH+H + and is the rate-limiting enzyme in guanosine monophosphate (GMP) synthesis, is amplified in tumors and rapidly proliferating tissues (Jackson et al, 1975) was an impetus for the development of effective anticancer, immunosuppressive and antiviral chemotherapies (Chen and Pankiewicz, 2007;Nair and Shu, 2007;Ratcliffe, 2006;Shu and Nair, 2008). Later studies showed that, when cells are treated with IMPDH inhibitors or when they are deprived of nutrients required for GMP synthesis, many cells form distinctive structures termed 'rods and rings' (RRs), mainly within the cytoplasm (Calise et al, 2014b;Carcamo et al, 2011;Gunter et al, 2008;Ji et al, 2006;Thomas et al, 2012). These macromolecular assemblies take the shape of rods that are typically 3-10 µm in length or rings that most often are 2-5 µm in diameter.…”

“…tubulin, actin and vimentin) Liu, 2010). Recent reports have demonstrated that RRs form in both the nucleus and cytoplasm (Calise et al, 2014b;Carcamo et al, 2014;Gou et al, 2014).…”