Comparable experiments in human osteosarcoma U2OS cells also showed that NAM treatment enhanced SHMT2 acetylation (Fig.?1a). is usually correlated Garcinol with poorer postoperative overall survival. Our study reveals the unknown mechanism of SHMT2 regulation by acetylation which is usually involved in colorectal carcinogenesis. Introduction One-carbon metabolism not only provides cellular components including nucleotides, lipids and proteins for cell growth but also generates glutathione and S-adenosylmethionine, which are needed to maintain the cellular redox status and epigenetic status of cells1. The role of one-carbon metabolism in tumorigenesis has been extensively analyzed2C4, and the antagonism of one-carbon metabolic enzymes has been used in chemotherapy for over 60 years5. Serine and glycine, two nonessential amino acids, are major inputs Garcinol for one-carbon metabolism and are utilized for nucleotide synthesis. Recently, disorders of serine and glycine metabolism during carcinogenesis have gained attention6. A key serine/glycine conversion enzyme whose expression is usually consistently altered during tumorigenesis is usually serine hydroxylmethyltransferase (SHMT). SHMT is the enzyme that catalyzes the reversible conversion of serine to glycine via the Garcinol transfer of the -carbon of serine to tetrahydrofolate (THF), and this conversion resulting in the formation of 5,10-methylene-THF and glycine; these in turn are involved in the folate cycle. Two SHMT genes, SHMT1 and SHMT2, have been recognized in the human genome. SHMT1 encodes the cytoplasmic isozyme involved in the de novo synthesis of thymidylate7, while SHMT2, which encodes the mitochondrial isozyme, participates in the synthesis of mitochondrial thymidine monophosphate (dTMP)8. Strikingly, SHMT2 but not SHMT1 expression is usually significantly upregulated in a variety of cancers, including colorectal, brain, central nervous system (CNS), kidney, and bladder cancers9,10. Two clinical studies have shown that high expression of SHMT2 is usually associated with tumor aggressiveness and prognosis11,12. In breast cancer, HIF1 and MYC cooperate to drive SHMT2 upregulation, which leads to an increased concentration of nicotinamide adenine dinucleotide phosphate (NADPH) and enhanced redox balance; this in turn facilitates cancer cell growth under hypoxic conditions10. However, whether post-translational modification affects the level of SHMT2 protein in tumorigenesis and how the upregulation of SHMT2 is usually involved in colorectal carcinogenesis are unknown. Two protein lysine modifications, acetylation and ubiquitination, are coordinately regulated to control critical cellular functions. Several metabolic enzymes are regulated by acetylation through ubiquitin-dependent proteasome degradation or lysosomal-dependent degradation13. In this study, we report that the activity and protein stability of the mitochondrial metabolic enzyme SHMT2 are regulated by lysine acetylation. Specifically, acetylation of lysine K95 inhibits SHMT2 activity and promotes K63-Ub-lysosome-dependent degradation of SHMT2 via macroautophagy. We investigated the functional significance of SHMT2 expression and acetylation levels in colorectal tumorigenesis. Our study reveals the previously unknown mechanism of SHMT2 regulation by acetylation in the one-carbon metabolic pathway that is involved in colorectal carcinogenesis. Results SHMT2 Garcinol is usually acetylated at K95 Recent mass spectrometry-based proteomic analyses have identified a large number of potentially acetylated proteins, including SHMT214. To confirm the acetylation of SHMT2 in vivo, Flag-tagged SHMT2 was ectopically expressed in HeLa cells and immunoprecipitated. Western blot with an anti-pan-acetyl-lysine antibody confirmed that SHMT2 was indeed acetylated and that its acetylation was enhanced approximately two-fold after treatment with nicotinamide (NAM, an inhibitor of the sirtuin (SIRT) family of deacetylases)15 (Fig.?1a). Comparable experiments in human osteosarcoma U2OS cells also showed that NAM treatment enhanced SHMT2 acetylation (Fig.?1a). In one of our previously published papers, we reported that acetylation at K464 of SHMT2 was increased by 4.7-fold in MEFs compared with MEFs14. In Garcinol addition, K280 in the catalytic domain name of SHMT2 was identified by an acetylation proteomics study16. To test whether these two sites are primary acetylation sites, we generated Arg (to mimic deacetyl-modification) and Gln (to mimic acetyl modification)17C19 substitution mutants of both sites (K280R, K280Q, K464R, K464Q). However, none of the mutants influenced the overall acetylation level of SHMT2 (Supplementary Fig.?1a), which indicates that neither K464 nor K280 is the major acetylation site of SHMT2 in our study. Moreover, the SHMT2 K464R/Q mutant exhibited an activity similar to that of the wild-type (WT) protein, while the K280R/Q mutant exhibited no activity due to Mouse monoclonal to Pirh2 disruption of the active site, which also suggests that our method of detection of SHMT2 activity is usually feasible (Supplementary Fig.?1b). To investigate the functional acetylated regulatory sites of SHMT2, mass spectrometry analysis was performed using Flag-tagged SHMT2-expressing stable cells. Lys95 of SHMT2 was found to be acetylated (Fig.?1b). Lys95 in SHMT2 is usually highly conserved in different species from to.