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Abstract The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the ‘glutamine addiction’ of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an ‘activation loop’ to assume a specific conformation that works together with a ‘lid’ to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression. 

Tumor-initiating cells contained within the aggressive brain tumor glioma (glioma stem cells, GSCs) promote radioresistance and disease recurrence. However, mechanisms of resistance are not well understood. Herein, we show that the proteome-level regulation occurring upon radiation treatment of several patient-derived GSC lines predicts their resistance status, whereas glioma transcriptional subtypes do not. We identify a mechanism of radioresistance mediated by the transfer of the metabolic enzyme NAMPT to radiosensitive cells through microvesicles (NAMPT-high MVs) shed by resistant GSCs. NAMPT-high MVs rescue the proliferation of radiosensitive GSCs and fibroblasts upon irradiation, and upon treatment with a radiomimetic drug or low serum, and increase intracellular NAD(H) levels. Finally, we show that the presence of NAMPT within the MVs and its enzymatic activity in recipient cells are necessary to mediate these effects. Collectively, we demonstrate that the proteome of GSCs provides unique information as it predicts the ability of glioma to resist radiation treatment. Furthermore, we establish NAMPT transfer via MVs as a mechanism for rescuing the proliferation of radiosensitive cells upon irradiation. 

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV–vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.