ABSTRACT
The p53-induced protein TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] is considered to be a F26BPase (fructose-2,6-bisphosphatase) with an important role in cancer cell metabolism. The reported catalytic efficiency of TIGAR as an F26BPase is several orders of magnitude lower than that of the F26BPase component of liver or muscle PFK2 (phosphofructokinase 2), suggesting that F26BP (fructose 2,6-bisphosphate) might not be the physiological substrate of TIGAR. We therefore set out to re-evaluate the biochemical function of TIGAR. Phosphatase activity of recombinant human TIGAR protein was tested on a series of physiological phosphate esters. The best substrate was 23BPG (2,3-bisphosphoglycerate), followed by 2PG (2-phosphoglycerate), 2-phosphoglycolate and PEP (phosphoenolpyruvate). In contrast the catalytic efficiency for F26BP was approximately 400-fold lower than that for 23BPG. Using genetic and shRNA-based cell culture models, we show that loss of TIGAR consistently leads to an up to 5-fold increase in the levels of 23BPG. Increases in F26BP levels were also observed, albeit in a more limited and cell-type dependent manner. The results of the present study challenge the concept that TIGAR acts primarily on F26BP. This has significant implications for our understanding of the metabolic changes downstream of p53 as well as for cancer cell metabolism in general. It also suggests that 23BPG might play an unrecognized function in metabolic control.
Subject(s)
Glycolates/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Phosphoric Monoester Hydrolases/chemistry , 2,3-Diphosphoglycerate/chemistry , Animals , Apoptosis Regulatory Proteins , Glycolates/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Muscle, Skeletal/enzymology , Phosphoric Monoester Hydrolases/metabolism , Recombinant Proteins/chemistry , Substrate Specificity , Transcription, GeneticABSTRACT
The regulation of synthesis, degradation, and distribution of lipids is crucial for homeostasis of organisms and cells. The sterol regulatory element-binding protein (SREBP) transcription factor family is post-translationally activated in situations of reduced lipid abundance and activates numerous genes involved in cholesterol, fatty acid, and phospholipid synthesis. In this study, we provide evidence that the primary transcript of SREBP2 contains an intronic miRNA (miR-33) that reduces cellular cholesterol export via inhibition of translation of the cholesterol export pump ABCA1. Notably, miR-33 also inhibits translation of several transcripts encoding proteins involved in fatty acid ß-oxidation including CPT1A, HADHB, and CROT, thereby reducing fatty acid degradation. The genetic locus encoding SREBP2 and miR-33 therefore contains a protein that increases lipid synthesis and a miRNA that prevents export and degradation of newly synthesized lipids. These results add an additional layer of complexity to our understanding of lipid homeostasis and might open possibilities for future therapeutic intervention.