Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism

Abstract

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.

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“…Involved in maintaining the homeostasis of cellular nucleotides by catalyzing the interconversion of nucleoside phosphates. AK3 knockout decreases proliferation and ATP levels in HeLa cells [22] ATPase H+ Transporting V1 Subunit C1 ATP6V1C1…”

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“…Although AK3 expression is low in pancreatic beta cells because they do not consume GTP, which is required for insulin secretion [ 60 ], its definite function has not been clarified. Recently, AK3 knockout in HeLa cells has been reported to decrease intracellular ATP levels; however, knockout mice studies are lacking and must be the direction of future research [ 61 ].…”

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“…Tumor cells are generally believed to consume high amounts of glucose to produce high energy levels [6]. In recent years, the relationship between cancer and glucose metabolism has attracted attention, and enzymes related to glycolysis and the tricarboxylic acid (TCA) cycle have been reported to be involved in cancer development and progression [7,8]. Metabolites or enzymes of the TCA cycle, such as fumarate hydratase (FH), isocitrate dehydrogenase, succinate dehydrogenase, and α-ketoglutarate dehydrogenase, are known to mutate or be deregulated in human cancers [9].…”