Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

Micronutrients from the diet and gut microbiota are essential to human health and wellbeing. Arguably, among the most intriguing and enigmatic of these micronutrients is queuine, an elaborate 7-deazaguanine derivative made exclusively by eubacteria and salvaged by animal, plant and fungal species. In eubacteria and eukaryotes, queuine is found as the sugar nucleotide queuosine within the anticodon loop of transfer RNA isoacceptors for the amino acids tyrosine, asparagine, aspartic acid and histidine. The physiological requirement for the ancient queuine molecule and queuosine modified transfer RNA has been the subject of varied scientific interrogations for over four decades, establishing relationships to development, proliferation, metabolism, cancer, and tyrosine biosynthesis in eukaryotes and to invasion and proliferation in pathogenic bacteria, in addition to ribosomal frameshifting in viruses. These varied effects may be rationalized by an important, if ill-defined, contribution to protein translation or may manifest from other presently unidentified mechanisms. This article will examine the current understanding of queuine uptake, tRNA incorporation and salvage by eukaryotic organisms and consider some of the physiological consequence arising from deficiency in this elusive and lesser-recognized micronutrient.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.

TRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr); an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle.

Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F₁F_O-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.

Eukaryotic life benefits from—and ofttimes critically relies upon—the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5′-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.