In a recent paper published in PNAS 109, 1092 (2012)*Alexandre Cassago et al. present a collection of results, ranging from tissue and cell culture studies to x-ray crystallography and biochemistry, that collectively suggest that the lesser studied of the three so far identified mammalian glutaminases, GAC, is the key glutaminase isozyme to supply for the increased tumor metabolic needs.
Though at very distinct rates, all cells in a living body grow and multiply. This process, called proliferation, must be tightly regulated in order for a healthy tissue – an organ like the lungs for example – to maintain its correct size and function. This if often accomplished through a carefully controlled event of production and release of proliferation-promoting signals, such as hormones, within a cell and its neighbors.
In order for a eukaryotic cell like ours to reproduce, it must duplicate its macromolecular biomass (genome, proteins, cell membrane) before they go ahead and divide. The sugar glucose is one of the main nutrients that will fuel this progression. Through a series of multistep processes performed by proteins generally termed metabolism, glucose is broken down and/or condensed into new molecules that will result in the generation of both energy (in form of ATP) and the synthesis of building blocks (nucleic acids, other amino acids, lipids) for the assembly of the macromolecules mentioned above.
Tumors are often a big ugly-looking deformed mass of cells that looks nothing like the organ it was removed from. Oncologists know, for quite some time now, that this is in great part due to the deregulation of the proliferation process. Cancer cells grow at their own will, at faster rates and at the expense of everything in its surroundings, like parasites. Therefore, cancer is generally defined as a group of diseases characterized by uncontrolled growth and spread of abnormal cells.
In order to provide for this seemingly uncontrolled growth, the energetic and biosynthetic metabolism of cancer cells ought to be readjusted. Tumors must then capture great amounts of other extracellular nutrients, such as the amino acid glutamine, and quickly and efficiently metabolize them for shunting into the appropriate pathways.
Glutamate production by mitochondrial glutaminase (GA), the first enzyme in glutaminolysis, is a key process for body homeostasis, and a crucial carbon donor for amino acid and lipid synthesis in tumor cells. To date, three GAs have been identified in humans: the Liver-type (or simply LGA), the Kidney-type (or KGA) and Glutaminase C (GAC), a splice variant of KGA (both usually referred to as GLS1).
In this extensive study, the authors present a collection of results, ranging from tissue and cell culture studies to x-ray crystallography and biochemistry, that collectively demonstrate that the lesser studied of the three so far identified mammalian glutaminases, Glutaminase C is the better adapted isozyme to supply for the increased tumor metabolic needs.
They believe that a clear distinction of the molecular and structural specifics of the three isozymes, especially between the two isoforms encoded by the gene gls, Glutaminase C and Kidney-type Glutaminase, is mandatory in the context of both understanding the mitochondrial glutamine-based metabolism of cancer and the future development of target-specific therapeutics.
In order to contribute to this distinction, they have introduced a number of original observations. They first show that although protein levels of the two kidney-type isoforms are increased in tumor tissues versus normal, only Glutaminase C is compartmentalized in the mitochondria, where glutaminolysis takes place. This is indeed surprising, as they both contain the canonical sequence that targets to the mitochondria. It seems this is the first study in the literature where isoform-specific antibodies are used exploring their unique C-termini, which could explain these new findings.
It has been known for several decades now that activity levels of the mammalian glutaminases respond to the presence of inorganic phosphate, though it is the first time that the three known isozymes are comprehensively studied together. The authors performed kinetic analysis of the three, and the outcome clearly shows that Glutaminase C most responsive to increasingly concentrations of the activator inorganic phosphate. This might be of particular importance in the context of glutaminolytic rates in tumors since hypoxic conditions may result in accumulation of this ion the mitochondria.
Furthermore, by solving Glutaminase C crystal structure in three different states, i.e., ligand-free, and either bound to L-glutamate or inorganic phosphate, they offer the structural basis for protein tetramerization-induced lifting of a “gating loop” as essential for the phosphate-dependent activation process. Amongst several new structural observations, they show that phosphate binds inside the catalytic pocket rather than the oligomerization interface, resulting in allosteric stabilization of tetramers and at the same time mediating substrate entry by competing with glutamate, therefore guaranteeing enzyme cycling. A higher tendency to oligomerize differentiates GAC from its splicing isoform and phosphate cycling in and out of the active site tells GAC specifically apart from the liver-type isozyme, known as inhibited by this ion. Besides, they believe the structural information will be particularly valuable in helping the future development of novel target-specific drug-based therapies to fight the aberrant cancer metabolism.
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