Structural and Biochemical Studies of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator)

Abstract

Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebra fish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate but not fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.The p53 tumor suppressor protein is a stress-induced transcription factor that controls various cellular response mechanisms including cell cycle arrest and apoptosis (1, 2). As part of a complex signaling network that includes positive and negative feedback loops, the p53 pathway determines cell death or survival after its activation by various forms of cellular damage (3). TIGAR (TP53-induced glycolysis and apoptosis regulator) is a recently identified p53-regulated gene that was found to lower fructose-2,6-bisphosphate (Fru-2,6-P 2 ) 2 levels in cells. TIGAR expression resulted in down-regulation of glycolysis, reduction of intracellular levels of reactive oxygen species, and protection from apoptosis (4).Fru-2,6-P 2 is a key regulator of cellular metabolism that activates glycolysis and inhibits gluconeogenesis. Fru-2,6-P 2 is found in almost all eukaryotes but is absent from prokaryotes (5). Other functions for this molecule in addition to its allosteric regulation of glycolysis and gluconeogenesis have been identified (6). The structure and function of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (FBPase-2) enzymes responsible for Fru-2,6-P 2 synthesis and removal within the cell have been studied for more than 20 years (7). The transcription of a hypoxia-inducible isoform (PFKFB3) is controlled by hypoxia-inducible factor 1 (8), and this isoform is frequently found in cancer cells (9). Thus, synthesis and removal of Fru-2,6-P 2 by PFKFB3 and TIGAR is controlled by hypoxia-inducible factor 1 and p53, emphasizing the importance of metabolic regulation for tumor suppression (10).Histidine phosphatases share a strictly conserved catalytic core centered at a histidine residue that is phosphorylated during the reaction. Two ...

Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy

“…In addition to the established role of p53 in cell cycle arrest and apoptosis, recent evidence has demonstrated that p53 reduces glycolytic flux and increases oxidative phosphorylation, halting the Warburg effect. [137][138][139][140][141][142][143] TIGAR (Tp53-induced glycolysis and apoptosis regulator) was identified to function as a fructose-2,6-bisphosphatase (Fru-2,6-BP), antagonizing the third step of glycolysis and thereby redirecting metabolite flux to the pentose phosphate pathway. [138][139][140] Interestingly, basal or low levels of p53 expression have been suggested to provide antioxidant effects and thus elicit cellular protection rather than cell death.…”

“…[137][138][139][140][141][142][143] TIGAR (Tp53-induced glycolysis and apoptosis regulator) was identified to function as a fructose-2,6-bisphosphatase (Fru-2,6-BP), antagonizing the third step of glycolysis and thereby redirecting metabolite flux to the pentose phosphate pathway. [138][139][140] Interestingly, basal or low levels of p53 expression have been suggested to provide antioxidant effects and thus elicit cellular protection rather than cell death. [138][139][140] A recent study has discovered an unexpected mechanism of TIGAR to decrease ROS generation at mitochondria.…”

“…[138][139][140] Interestingly, basal or low levels of p53 expression have been suggested to provide antioxidant effects and thus elicit cellular protection rather than cell death. [138][139][140] A recent study has discovered an unexpected mechanism of TIGAR to decrease ROS generation at mitochondria. 144 TIGAR, upon hypoxia, translocates to mitochondria and interacts with mitoHK-II, stabilizing mitoHK-II and thereby decreasing mitochondrial ROS generation and providing cellular protection.…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…While this work was in progress, two other crystal structures of Eco-ZnuA were reported [13,14] and are virtually identical in fold to the structure of ZnZnuA form I (Fig. 1).…”

“…6803 MntC (Syn-MntC) [12], and Eco-ZnuA [13,14] as well as the metal-free (apo) structures of Tpa-TroA [15] and mutant Syn-ZnuA (without the His-rich loop) [8] have been determined by X-ray crystallography. The overall architecture of these MBRs is similar to that of other PLBPs from ABC transporter systems.…”