Glutaminase Affects the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor γ (PPARγ) via Direct Interaction.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats.

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We 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 retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.