TIGAR: a p53 regulated enzyme with an effect on apoptosis and autophagy.

The gene TIGAR (Tp53-induced glycolytic and apoptotic regulator) has originally been described as a direct target gene of the tumor suppressor protein p53 (3). Based on sequence similarity, it was suggested that TIGAR would act on fructose 2,6-bisphosphate (F26BP), an important allosteric activator of one of the key-enzymes of glycolysis, phosphofructokinase-1 (4). As such, the activity of TIGAR could reduce F26BP levels and glycolytic flux when p53 is activated under conditions of cellular stress. Consistent with this hypothesis, Bensaad and colleagues showed that overexpression of TIGAR leads to a modest decrease of F26BP and reduces glycolytic flux (3). In reverse, shRNA mediated knock-down of TIGAR led to the opposite effect. Functionally, the inhibition of glycolysis led to reduced apoptosis and autophagy (3, 5). This was attributed to increased NADPH production in the pentose phosphate pathway and ensuing resistance to oxidative stress (Fig. 1a & b). Subsequently, the analysis of TIGAR knockout mice revealed a reduction of colorectal tumor formation (6, 7) and a partial protection from ischemic heart disease (8). Furthermore, downregulation of TigarB improved the phenotype in a zebrafish model of Parkinson’s disease (9).   

 

The physiologic substrate of TIGAR might still be elusive.

From a biochemical standpoint, these strong phenotypes were surprising since four different enzymes had already been known for a long time to catalyse the same reaction that TIGAR was postulated to perform (i.e. the degradation of fructose 2,6-bisphosphate (F26BP)). In fact, the four enzymes PFKFB1 to PFKFB4 are bifunctional proteins that combine a kinase that synthesizes F26BP with a phosphatase that degrades F26BP (10). The reciprocal regulation of these activities by many allosteric regulators has been thoroughly evaluated in the 1980s and is presented in most biochemical textbooks. Given that previous studies had not actually assessed the activity of TIGAR, we purified this enzyme and assessed its activity on F26BP, expecting to find a very high activity. Surprisingly, the fructose 2,6-bisphosphatase activity of TIGAR turned out to be extremely low (11, 12) – several order of magnitudes lower than the activity of the four enzymes that usually perform this reaction in cells (PFKFB1 to 4; comparing kcat/KM)(11). Yet, we identified several substrates that were metabolized up to 400x better than F26BP (e.g. 2,3-bisphosphoglycerate, 2,3-BPG) (11). Notably, all good substrates of TIGAR shared a remarkable similarity in their structure (with a phosphate group being attached adjacent to a carboxylic acid group), which was not shared with F26BP (11)(Fig. 1c). Furthermore, we found that TIGAR did not remove phosphate from carbon C2, which is normally removed by PFKFB1- 4, but rather removed the phosphate from carbon C6, suggesting that F26BP binds in a completely different orientation in the catalytic pocket of TIGAR than it does in PFKFB1 – 4 (11). This might seem an irrelevant detail at first sight. Yet, the lack of conservation of the orientation within the catalytic pockets is relevant, since it was the homology between TIGAR and PFKFB1-4 that had originally led to the proposition that TIGAR would act on F26BP(3).

Collectively, these results made it extremely unlikely that TIGAR had been maintained during evolution to directly regulate fructose-2,6-bisphosphate levels. Unfortunately, it was unclear how the accumulation of any of the better TIGAR substrates could lead to the changes in apoptosis, autophagy, reactive oxygen species (ROS) and F26BP levels, which had been described in TIGAR knockout or knockdown cells (3, 5) (Fig. 1d). Overall, we concluded that the physiological substrate of TIGAR and the mechanism of its action in cells might still be elusive.