mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis
A working model on regulation of GLDC activity by coordinated acetylation and polyubiquitination. GLDC is acetylated at K514 by ACAT1 following mTORC1 inhibition.
|核心内容：
甘氨酸脱羧酶(GLDC)将甘氨酸裂解转化为单碳单位，是甘氨酸裂解系统的关键酶。GLDC通常表达上调，并在许多人类癌症中发挥重要作用。 GLDC是否以及如何受到翻译后修饰的调控尚不清楚。 在这里，我们报道了雷帕霉素复合物1(mTORC1)信号通过诱导去乙酰化酶sirtuin3(SIRT3)的转录来抑制GLDC赖氨酸(K)514位点的乙酰化。 在mTORC1被抑制后，乙酰转移酶乙酰辅酶a乙酰转移酶1(ACAT1)催化GLDCK514乙酰化。GLDC的乙酰化损害了它的酶活性。 此外，GLDC的这种乙酰化启动了泛素连接酶NF-X1对K33连接的多泛素化，导致其被蛋白酶体途径降解。 最后，我们发现GLDCK514乙酰化抑制甘氨酸分解代谢、嘧啶合成和胶质瘤肿瘤发生。 我们的发现揭示了GLDC翻译后修饰在调节其酶活性、甘氨酸代谢和肿瘤发生中的关键作用，并为胶质瘤等癌症的治疗提供了潜在的靶点。Mechanistic target of rapamycin (mTOR) is a conserved serine–threonine kinase in the phosphoinositide-3 kinase-related kinase family, which integrates a wide array of extracellular and intracellular signals to regulate cell growth, metabolism, translation, and autophagy1–3. mTOR is the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). The rapamycin-FKBP12 complex directly inhibits mTORC1, whereas mTORC2 is insensitive to acute rapamycin treatment1. mTORC1 is defined by its three core components: mTOR, regulatory protein associated with mTOR (RPTOR), and mammalian lethal with Sec13 protein 8 (mLST8)4. Dysregulation of mTORC1 activity induces highly active cell metabolism and proliferation states, which is commonly observed in many human cancers, including glioblastoma (GBM)5,6. Glycine is a nonessential amino acid, which is an important residue of many proteins. Glycine can also be cleaved to yield one-carbon units, which are used for nucleotide synthesis via the tetrahydrofolate (THF) cycle5,7. In addition, high levels of glycine are toxic by its conversion to metabolites, such as aminoacetone and methylglyoxal8. The glycine cleavage system controls glycine catabolism through multi-step reactions, generating CO2, NH3, NADH, and 5,10-methylene-THF9,10. The glycine cleavage system is a multi-enzyme complex, consisting of glycine decarboxylase (GLDC, also called P protein), amino-methyltransferase (T protein), dihydrolipoamide dehydrogenase (L protein), and the hydrogen carrier protein (H protein)11. Various studies have demonstrated that glycine metabolism is essential for tumorigenesis7,8,12. GLDC is a mitochondrial pyridoxal 5’-phosphate (PLP)- dependent enzyme that catalyzes the first and rate-limiting step in glycine catabolism11. GLDC binds glycine through its PLP cofactor to form an external aldimine that loses the carboxyl group as CO2 and donates the remaining aminomethylene moiety to the oxidized lipoamide arm of H protein9. Mutations in GLDC gene cause glycine accumulation, leading to neural tube defect and glycine encephalopathy (also known as nonketotic hyperglycinemia)13,14. It has also been demonstrated that GLDC is hyperactive in different types of cancer cells and plays a fundamental role in tumor growth. For example, increased expression of GLDC in non-small cell lung cancer-initiating cells is essential for tumorigenesis by promoting pyrimidine biosynthesis, glycolysis, and sarcosine production15. GLDC expression is markedly increased in MYCN-amplified neuroblastomas, which is required for neuroblastoma cell proliferation and tumorigenicity16. In this study, we found that GLDC is acetylated at K514 by ACAT1 following mTORC1 inhibition. GLDC K514 acetylation inhibited its enzymatic activity, promoted its K33-linked polyubiquitination at K544 by NF-X1 and proteasomal degradation, and suppressed glioma tumor growth. Our findings suggest that GLDC activity is regulated by sequential posttranslational modifications, including acetylation and polyubiquitination, and reveal critical regulatory mechanisms of glycine metabolism and tumorigenesis. Results Inhibition of mTORC1 suppresses GLDC activity by promoting its acetylation at K514. It has been shown that mTORC1 regulates certain amino acid metabolism and tumorigenesis. However, whether it regulates GLDC-mediated glycine metabolism is unknown. We generated U251 glioma cells stably expressing Flag-tagged GLDC and treated the cells with the mTORC1 inhibitor Rapamycin or left them untreated. We then purified Flag-GLDC by anti-Flag immunoaffinity beads and measured its enzymatic activity. The results indicated that Rapamycin treatment suppressed GLDC enzymatic activity (Fig. 1a). We next explored whether GLDC activity is regulated by posttranslational modifications. We found that Rapamycin treatment inhibited phosphorylation of S6K and 4EBP1 (hallmarks of mTORC1 activation) but did not affect serine/threonine or tyrosine phosphorylation of GLDC (Supplementary Fig. 1a). Interestingly, immunoblotting analysis indicated that Rapamycin treatment increased GLDC acetylation (Fig. 1b). RPTOR is a core component of mTORC1. In RPTOR-deficient cells, the basal acetylation of GLDC was increased and Rapamycin treatment did not further increase its acetylation (Fig. 1c). These results suggest that mTORC1 signal inhibits GLDC acetylation. Analysis of proteomic databases indicates that 15 lysine residues of GLDC are potentially acetylated (https://www./ ). To test whether these residues are primary acetylation sites regulated by mTORC1, we generated Arg (R) (to mimic deacetylated lysine) substitution mutants of each lysine residue. We found that Rapamycin treatment induced increase of acetylation of wild-type GLDC and its 14 mutants but not the GLDCK514R mutant, which had basal acetylation similar as wildtype GLDC without Rapamycin treatment (Fig. 1d). These results suggest that, while GLDC is basally acetylated at other lysines, Rapamycin induces acetylation of GLDC at K514. Human GLDC K514 is conserved across species (Supplementary Fig. 1b). Previous studies have demonstrated that K514 is localized in the catalytic pocket of GLDC9,17. We generated an antibody specifically recognizing K514-acetylated GLDC (anti-Ac-K514- GLDC; Supplementary Fig. 1c). Immunoblotting analysis with this antibody confirmed that GLDC K514 was acetylated and this was increased following Rapamycin treatment (Fig. 1e, f). These results suggest that K514 is the major acetylation site negatively regulated by mTORC1. To investigate the effects of K514 acetylation of GLDC, we generated GLDCK514Q mutant, which mimics its K514 acetylation. We transfected Flag-tagged wild-type GLDC, GLDCK514R, and GLDCK514Q into HEK293 cells and purified these proteins by anti-Flag immunoaffinity beads. We found that the enzymatic activity of wild-type GLDC was inhibited in Rapamycin-treated cells. GLDCK514R had slightly reduced activity in comparison to wild-type GLDC, but its activity was not inhibited following rapamycin treatment. However, the enzymatic activity of GLDCK514Q was impaired in untreated and Rapamycin-treated cells (Fig. 1g). Taken together, these results suggest that inhibition of mTORC1 signal suppresses GLDC enzymatic activity by promoting its K514 acetylation.
