Friday, March 14, 2025
3/14/2025
PROJECT SUMMARY/ABSTRACT Mitochondrial glutaminases (GLS and GLS2) catalyze the hydrolysis of glutamine to glutamate and ammonium, a metabolic reaction that is critical for numerous aspects of mammalian physiology. Whereas GLS2 expression is largely restricted to the liver, GLS is ubiquitously expressed, with particularly high levels in the kidney, digestive tract, and brain. Its roles include the regulation of systemic acid-base homeostasis and the biosynthesis of excitatory and inhibitory neurotransmitters. Accordingly, human inborn errors of metabolism involving mutation of the GLS gene have severe phenotypes, including neural excitotoxicity and lethal neonatal encephalopathy. In addition to its functions in organismal health, dysregulated GLS activity has been implicated in a spectrum of human pathologies, ranging from neurodegeneration to cancer. Consequently, there have been intensive efforts to develop selective allosteric inhibitors of GLS, one of which is now being evaluated in clinical trials for the treatment of solid and hematological malignancies. However, results to date indicate that the rapid development of resistance limits the efficacy of this therapeutic strategy. Remarkably, despite the long history of GLS research, it is not understood how the activity of this enzyme is regulated in cells. Purified recombinant GLS has minimal catalytic activity, and vastly supraphysiological concentrations of inorganic phosphate (100-150 mM) are required to activate GLS in vitro. We have recently discovered that that the bioactive phospholipid phosphatidic acid (PA) is an extremely potent activator of GLS, approximately 107-fold more potent than inorganic phosphate. Importantly, we have found that PA can activate GLS even in the presence of clinical GLS inhibitors, thus rendering these drugs ineffective. In this project, we propose to define the molecular mechanism by which PA activates GLS, and then to probe the upstream regulation of mitochondrial PA signaling, with the ultimate goal of perturbing this process to overcome resistance to GLS inhibitors. In the course of these studies, we will develop new chemical probes for investigating the PA-GLS interaction, including phosphorus-based covalent capture probes to determine the binding site of the PA phosphomonoester head group. We will then conduct a screen to identify additional potential metabolite-GLS interactions, and functionally validate any ‘hits’ obtained. In the second Aim, we will build on preliminary data that strongly implicate the PA-generating enzyme phospholipase D2 (PLD2), as well as the uncharacterized protein PLD5, in the regulation of the mitochondrial PA-GLS axis. We will attempt to determine the function of PLD5, focusing on whether its reported interaction with PLD2 regulates either the activity or subcellular localization of the latter. Finally, since our preliminary data indicate that PLD2/5 mediate resistance to clinical GLS inhibitors, we will test whether pharmacological or genetic blockade of PLD2 or PLD5 can overcome this resistance mechanism. Thus, our proposed studies will define the physiological processes that regulate GLS activity, and will then apply this knowledge to identify opportunities for enhancing the efficacy of therapeutic strategies targeting glutamine metabolism.
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