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GART trifunctional enzyme: Biological Overview | References |

Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).

Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).

Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).

To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met²⁷, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).

The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (Tiong, 1990). Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).

In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).

CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim⁰¹, Cry⁰¹, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).

Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).

The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).

This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).

Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).

This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).

Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera

The Drosophila Gart locus consists of two genes. One gene encodes three enzymes in the de novo purine nucleotide biosynthesis pathway [glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART)]. The second gene lies within an intron of the purine gene and encodes a cuticle protein. To investigate the evolution of the Gart locus, the Chironomus tentans homolog was cloned by screening a genomic DNA library with a polymerase chain reaction product. This study shows that the interesting structural features of this locus conserved in two distant Drosophila species are not found in the Chironomus homolog. These features include the cuticle protein gene nested within an intron and the existence of an alternative transcript to yield a monofunctional enzyme. In addition, the extremely rapid divergence of coding sequence seen for members of the tandemly duplicated AIRS domain in Drosophila is found to be much less rapid in Chironomus (Clark, 1992).

Genetic analysis of the adenosine3 (Gart) region of the second chromosome of Drosophila melanogaster

The Gart gene of Drosophila melanogaster is known, from molecular biological evidence, to encode a polypeptide that serves three enzymatic functions in purine biosynthesis. It is located in polytene chromosome region 27D. One mutation in the gene (ade3¹) has been described previously. This study report forty new ethyl methanesulfonate-induced mutations selected aga!nst a synthetic deficiency of the region from 27C2-9 to ++28B3-4. The mutations were characterized cytogenetically and by complementation analysis. The analysis apparently identifies 12 simple complementation groups. In addition, two segments of the chromosome exhibit complex complementation behavior. The first, the 28A region, gave three recessive lethals and also contains three known visible mutants, spade (spd), Sternopleural (Sp) and wingless (wg); a complex pattern of genetic interaction in the region incorporates both the new and the previously known mutants. The second region is at 27D, where seven extreme semilethal mutations give a complex complementation pattern that also incorporates ade3¹. Since ade3¹ is defective in one of the enzymatic functions encoded in the Gart gene, it is assumed that the other seven also affect the gene. The complexity of the complementation pattern presumably reflects the functional complexity of the gene product. The phenotypic effects of the mutants at 27D are very similar to those described for ade2 mutations, which also interrupt purine biosynthesis (Tiong, 1990).

Conserved arrangement of nested genes at the Drosophila Gart locus

The Drosophila melanogaster Gart gene encodes three enzymatic activities in the pathway for purine de novo synthesis. Alternative processing of the primary transcript leads to the synthesis of two overlapping polypeptides. The coding sequence for both polypeptides is interrupted by an intron that contains a functional cuticle protein gene encoded on the opposite DNA strand. This nested organization also exists at the homologous locus of a distantly related species, Drosophila pseudoobscura. In both species, the intronic cuticle gene is expressed in wandering larvae and in prepupae. Remarkably, there are 24 different highly conserved noncoding segments within the intron containing the cuticle gene. These are found upstream of the transcriptional start, at the 3' end, and even within the single intronic gene intron. Other introns in the purine gene, including the intron at which alternative processing occurs, show no such homologies. It seems likely that at least some of the conserved noncoding regions are involved in specifying the high level developmental expression of the cuticle gene. Shared cis-acting regulatory sites might enhance transcription of both genes and help explain their nested arrangement (Henikoff, 1987).

GART Functions as a Novel Methyltransferase in the RUVBL1/beta-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133(+) cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/beta-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/beta-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC (Tang, 2023).

Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods

Hematopoietic stem cells (HSCs) are indispensable for the maintenance of the entire blood program through cytokine response. However, HSCs have high radiosensitivity, which is often a problem during radiation therapy and nuclear accidents. Although a previous study has reported that the combination cytokine treatment (interleukin-3, stem cell factor, and thrombopoietin) improves the survival of human hematopoietic stem/progenitor cells (HSPCs) after radiation, the mechanism by which cytokines contribute to the survival of HSPCs is largely unclear. To address this issue, the present study characterized the effect of cytokines on the radiation-induced gene expression profile of human CD34(+) HSPCs and explored the hub genes that play key pathways associated with the radiation response using a cDNA microarray, a protein-protein interaction-MCODE module analysis and Cytohubba plugin tool in Cytoscape. This study identified 2,733 differentially expressed genes (DEGs) and five hub genes (TOP2A, EZH2, HSPA8, GART, HDAC1) in response to radiation in only the presence of cytokines. Furthermore, functional enrichment analysis found that hub genes, genes that have many interactions with other genes, and top DEGs based on fold change were enriched in the chromosome organization and organelle organization. The present findings may help predict the radiation response and improve understanding of this response of human HSPCs (Sato, 2023).

GART expression in rat spinal cord after injury and its role in inflammation

The glycinamide ribonucleotide transformylase (GART) gene, a trifunctional polypeptide, has phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase activity, and is required for de novo purine biosynthesis. GART is highly conserved in vertebrates. Alternative splicing of GART results in two transcript variants encoding different isoforms. However, the expression and function of GART in the central nervous system lesion are still unclear. This study used a traumatic spinal cord injury (SCI) model in adult Sprague-Dawley rats and investigated the dynamic changes of GART protein expression in the spinal cord. Western blot analysis revealed that GART was present in sham-operated spinal cord. It gradually increased, reached a peak at day 3 after SCI, and then declined during the following days. Double immunofluorescence staining revealed a widespread of GART, and the majority of GARTs are detected in astrocytes. After injury, GART expression was increased predominantly in astrocytes, positively correlated with the highly expressed proliferating cell nuclear antigen (PCNA). Knockdown of GART expression in cultured primary astrocytes by siRNA revealed that expression of GART in astrocytes plays a role in the LPS-induced release of pro-inflammatory factors, such as TNF-alpha and IL-6. These results showed that GART may participate in the pathophysiology of SCI, and more research is needed to have a good understanding of its function and mechanism (Zhang, 2014).

Search PubMed for articles about Drosophila Gart

Clark D. V., Henikoff, S. (1992). Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera. J Mol Evol. 35(1):51-59. PubMed ID: 1518084

He, L., Wu, B., Shi, J., Du, J. and Zhao, Z. (2023). Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila. Cell Rep 42(8): 112912. PubMed ID: 37531254

Henikoff, S. and Eghtedarzadeh, M. K. (1987). Conserved arrangement of nested genes at the Drosophila Gart locus. Genetics 117(4):711-725. PubMed ID: 3123310

Tiong S. Y. and Nash, D. (1990). Genetic analysis of the adenosine3 (Gart) region of the second chromosome of Drosophila melanogaster. Genetics 124(4):889-897. PubMed ID: 2108904

Sato Y., Yoshino, H., Ishikawa, J., Monzen, S., Yamaguchi, M., Kashiwakura, I. (2023). Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods. Sci Rep13(1):10762. PubMed ID: 37402866

Tang, C., Ke, M., Yu, X., Sun, S., Luo, X., Liu, X., Zhou, Y., Wang, Z., Cui, X., Gu, C. and Yang, Y. (2023). GART Functions as a Novel Methyltransferase in the RUVBL1/beta-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer. Adv Sci (Weinh)10(25):e2301264. PubMed ID: 37439412

Zhang, D., Yue, Y., Jiang, S., Li, A., Guo, A., Wu, X., Xia, X., Cheng, H., Zhang, J., Tao, T., Gu, X. (2014). GART expression in rat spinal cord after injury and its role in inflammation. Brain Res 1564:41-51. PubMed ID: 24709117

date revised: 9 December 2023

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