ATP citrate lyase ACLY is a
ATP citrate lyase (ACLY) is a cytosolic enzyme that catalyzes the generation of acetyl-CoA from citrate . Acetyl-CoA is the building block for the biosynthesis of fatty acids and cholesterol, and also serves as a substrate of the acetylation reaction of various molecules, including histones and polyamines , , . Lipid anabolism and histone acetylation are generally associated with cell proliferation, and indeed ACLY is activated in various types of cancer . The inhibition of ACLY induces proliferation arrest in cancer Evans Blue tetrasodium salt synthesis , . Although ACLY is mainly regulated at the transcriptional level , it is also known that ACLY is degraded by the 26S proteasome and ubiquitination and acetylation of the same lysine residue determines the stability of ACLY . In addition to these quantitative regulation mechanisms, ACLY is activated by phosphorylation at Ser-455, which is catalyzed by PI3K/Akt pathway .
Material and methods
Discussion Polyamines are essential for cell proliferation and their concentrations are generally high in cancer cells. ACLY is also related to cell proliferation and is activated in many types of cancer . However, the relationship between polyamine metabolism and ACLY has remained unaddressed. In this study, we demonstrated that AZ, a polyamine-induced negative regulator of polyamines, binds to ACLY. ACLY was co-immunoprecipitated by both AZ1 and AZ2, and colocalized to the cytoplasm with both AZ1 and AZ2 (Fig. 1A, B). It is known that AZ binds to ODC to accelerate the degradation of ODC protein by the 26S proteasome without ubiquitination. It has also been reported that other AZ1-binding proteins such as Smad1, cyclin D1 and Aurora-A are destabilized by AZ1 , , . We had hypothesized that binding of AZ would accelerate the degradation of ACLY; however, the stability of ACLY was hardly affected by AZ-binding (Fig. 2). We then demonstrated in vitro that both AZ1 and AZ2 enhance ACLY activity in a dose-dependent manner (Fig. 3A). In the cancer cells, putrescine, an inducer of endogenous AZ, increased the activity of ACLY (Fig. 3B). The increase in ACLY activity by putrescine was inhibited by AZ1 knockdown and partially inhibited by AZ2 knockdown, indicating that AZ directly enhances the activity of ACLY, likely through a protein–protein interaction. The molecular mechanism for the activation of ACLY by AZ is of great interest and should be studied in future investigations. Knockdown of AZ1 or AZ2 in putrescine-untreated cells significantly decreased the ACLY activity (Fig. 4A). Thus, ACLY should be partially activated by endogenous AZ in the cells under physiological conditions. The reduction in ACLY activity was greater following AZ1 knockdown than AZ2 knockdown. This is likely because the expression of AZ1 mRNA in mammalian cells is much higher than that of AZ2 mRNA . Our results also indicated that the intracellular level of AZ1 mRNA was 13-fold and 7-fold higher in LNCaP cells and A549 cells, respectively, than that of AZ2 mRNA (data not shown). Under the same condition, the knockdown of AZ1 or AZ2 significantly decreased the cellular levels of acetyl-CoA, a product of ACLY (Fig. 4B). In A549 cells, AZ knockdown also lowered the cellular level of cholesterol, which is synthesized from acetyl-CoA, although the decrease was slight in LNCaP cells. The reason for this difference is unclear, however, lipid metabolism might be quantitatively different between the two cancer cell lines. Polyamines are basic molecules, and spermidine and spermine in particular bind to cellular acidic molecules, such as nucleic acid. In proliferating cells, such acidic molecules increase and more polyamines are required. Meanwhile, excessive concentrations of polyamines are cytotoxic, inducing cell death, thus cells are equipped with feedback regulatory mechanisms for polyamines. AZ suppresses both polyamine synthesis and the uptake of extracellular polyamines. Cellular spermidine and spermine induce spermidine/spermine-N1-acetyltransferase (SSAT), which acetylates polyamines for further catabolism by acetylpolyamine oxidase or excretion from the cells , . In SSAT transgenic mice, cytosolic acetyl-CoA is depleted by the acetylation of spermidine and spermine, leading to a decreased synthesis of fatty acid and lean phenotype . Thus, in cells with high spermidine and spermine concentrations, the cytosolic acetyl-CoA requirement must be higher. It is possible that the significance for enhancing the ACLY activity by AZ is to provide acetyl-CoA as the substrate of SSAT. Intracellular concentration of acetyl-CoA has been shown to be relevant to diverse cellular activities including cell proliferation .