mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.

Stand-alone lipoylated H-protein of the glycine cleavage system enables glycine cleavage and the synthesis of glycine from one-carbon compounds in vitro

H-protein, one of the four component proteins (H, T, P and L) of glycine cleavage system (GCS), is generally considered a shuttle protein interacting with the other three GCS-proteins via a lipoyl swinging arm. We report that without P-, T- and L-proteins, lipoylated H-protein (Hlip) enables GCS reactions in both glycine cleavage and synthesis directions in vitro. This apparent catalytic activity is closely related to the cavity on the H-protein surface where the lipoyl arm is attached. Heating or mutation of selected residues in the cavity destroys or reduces the stand-alone activity of Hlip, which can be restored by adding the other three GCS-proteins. Systematic study of the Hlip-catalyzed overall GCS reactions and the individual reaction steps provides a first step towards understanding the stand-alone function of Hlip. The results in this work provide some inspiration for further understanding the mechanism of the GCS and give some interesting implications on the evolution of the GCS.

Glycine Cleavage System H Protein Is Essential for Embryonic Viability, Implying Additional Function Beyond the Glycine Cleavage System

Glycine cleavage system H protein (GCSH) is a component of the glycine cleavage system (GCS), a conserved protein complex that acts to decarboxylate glycine. Mutation of AMT or GLDC, encoding the GCS components aminomethyltransferase and glycine decarboxylase, can cause malformations of the developing CNS (neural tube defects (NTDs) and ventriculomegaly) as well as a post-natal life-limiting neurometabolic disorder, Non-Ketotic Hyperglycinemia. In contrast, it is unclear whether mutation of GCSH contributes to these conditions and we therefore investigated GCSH loss of function in mice. Mice that were heterozygous for a Gcsh null allele were viable and did not exhibit elevated plasma glycine. Moreover, heterozygous mutation of Gcsh did not increase the frequency of NTDs in Gldc mutant embryos. Homozygous Gcsh null mice were not recovered at post-natal stages. Analysis of litters at E8.5-10.5, revealed the presence of homozygous null embryos which were much smaller than littermates and had failed to develop beyond early post-implantation stages with no visible somites or head-folds. Hence, unlike null mutations of Gldc or Amt, which are compatible with embryonic survival despite the presence of NTDs, loss of Gcsh causes embryonic death prior to mid-gestation. Maternal supplementation with formate did not restore embryonic development beyond E7.5, suggesting that the primary cause of lethality was not loss of glycine cleavage activity or suppression of folate one-carbon metabolism. These findings suggest that GCSH has additional roles beyond function in the glycine cleavage system. We hypothesize that GCSH potentially acts in lipoylation of 2-oxoacid dehydrogenase proteins, as reported in bacteria.

Genomic Insights Into the Lifestyles of Thaumarchaeota Inside Sponges

Sponges are among the oldest metazoans and their success is partly due to their abundant and diverse microbial symbionts. They are one of the few animals that have Thaumarchaeota symbionts. Here we compare genomes of 11 Thaumarchaeota sponge symbionts, including three new genomes, to free-living ones. Like their free-living counterparts, sponge-associated Thaumarchaeota can oxidize ammonia, fix carbon, and produce several vitamins. Adaptions to life inside the sponge host include enrichment in transposases, toxin-antitoxin systems and restriction modifications systems, enrichments previously reported also from bacterial sponge symbionts. Most thaumarchaeal sponge symbionts lost the ability to synthesize rhamnose, which likely alters their cell surface and allows them to evade digestion by the host. All but one archaeal sponge symbiont encoded a high-affinity, branched-chain amino acid transporter system that was absent from the analyzed free-living thaumarchaeota suggesting a mixotrophic lifestyle for the sponge symbionts. Most of the other unique features found in sponge-associated Thaumarchaeota, were limited to only a few specific symbionts. These features included the presence of exopolyphosphatases and a glycine cleavage system found in the novel genomes. Thaumarchaeota have thus likely highly specific interactions with their sponge host, which is supported by the limited number of host sponge species to which each of these symbionts is restricted.

Structure-based dynamic analysis of the glycine cleavage system suggests key residues for control of a key reaction step

Molecular shuttles play decisive roles in many multi-enzyme systems such as the glycine cleavage system (GCS) for one-carbon (C1) metabolism. In GCS, a lipoate swinging arm containing an aminomethyl moiety is attached to protein H and serves as a molecular shuttle among different proteins. Protection of the aminomethyl moiety in a cavity of protein H and its release induced by protein T are key processes but barely understood. Here, we present a detailed structure-based dynamic analysis of the induced release of the lipoate arm of protein H. Based on molecular dynamics simulations of interactions between proteins H and T, four major steps of the release process showing significantly different energy barriers and time scales can be distinguished. Mutations of a key residue, Ser-67 in protein H, led to a bidirectional tuning of the release process. This work opens ways to target C1 metabolism in biomedicine and the utilization of formate and CO2 for biosynthesis.

Correction to: Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

An amendment to this paper has been published and can be accessed via the original article.

Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.