Evolutionary, structural and mechanistical analyses of the multidomain architecture of the human glutaminases and study of the interaction between glutaminases and the peroxisome proliferator-activated receptor gamma

On the basis of the detection of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, 2 human genes are known to encode at least 4 glutaminase isoforms. In this work, we investigated the origin and evolution of the glutaminases. We built a phylogenetic tree, and propose that the multidomain architecture of glutaminases is a feature inherited from bacterial ancestors. We also propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retro transposon event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in the evolutionary process. To obtain information on ANK folding, we solved the high-resolution structure of the ANK repeat- containing C-termini of kidney-type glutaminase (KGA). We also solved the novel crystal structure of multidomain KGA, containing N-terminal, glutaminase and C-terminal domains. The structures explain these proteins¿ compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Considering the presence of protein-protein interaction domains, such as ANK and Nuclear Receptor (NR) boxes, we investigated the interaction of KGA with other proteins. The Peroxisome Proliferator-Activated Receptor Gamma (PPAR?) was previously identified in our group as interaction partner of KGA using yeast two-hybrid approach. In this work, we determined that the Ligand Binding Domain (LBD) PPAR? and both N-terminal and glutaminase domains of KGA are sufficient for mediating the interaction. KGA showed a relatively low affinity with the receptor. The apparent dissociation constants experimentally calculated fall into micromolar range and are indicative of a weak and transient interaction. Moreover, non tumoral (HEK-293T) and tumoral (MDA-MB-436) cells overexpressing KGA showed an increase of the transcriptional activity of PPAR?. Collectively, these results provide information about glutaminases that will aid in the design of isoform-oriented glutaminase inhibitors (AU)