Thuy-Tien Thi Nguyen

All Publications

• A Generalizable Fluorescence Sensor Platform for Sample Preparation-Free Protein Detection. Advanced materials (Deerfield Beach, Fla.) Wu, H. D., Trinh, T., Li, T., Thapa, S., Lee, R. B., Nguyen, T. T., Petrakian, C. F., Eisenstein, M., Schneebeli, S. T., Li, J., Tom Soh, H. 2025: e19662

  Abstract

  Modern molecular detection assays such as enzyme-linked immunosorbent assays (ELISAs) offer excellent sensitivity and specificity, but typically require multiple reagents and extensive sample preparation, limiting their usefulness as rapid diagnostics. A generalizable biosensor platform is introduced that enables single-step, sample preparation-free detection of protein analytes with high sensitivity in complex samples. The NanoFluor system employs Janelia Fluor dyes coupled to a nanobody via HaloTag conjugation with a flexible glycine-serine linker, where the dye undergoes a switch from a non-fluorescent to a fluorescent state when the coupled nanobody binds to its target. It is demonstrated that the NanoFluor design achieves detection limits as low as picomolar concentrations across diverse protein targets. Molecular dynamics simulations, coupled with quantum mechanics/molecular mechanics computational models, reveal the mechanistic basis for the fluorescence change, and demonstrate the feasibility of multiplexed detection in complex samples including undiluted serum. This versatile, simple biosensor design can prove valuable for point-of-care diagnostics and other molecular detection applications.

  View details for DOI 10.1002/adma.202419662

  View details for PubMedID 40847924

• Filament formation drives catalysis by glutaminase enzymes important in cancer progression NATURE COMMUNICATIONS Feng, S., Aplin, C., Nguyen, T. T., Milano, S. K., Cerione, R. A. 2024; 15 (1): 1971

  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.

  View details for DOI 10.1038/s41467-024-46351-3

  View details for Web of Science ID 001179853600006

  View details for PubMedID 38438397

  View details for PubMedCentralID PMC10912226

• High-resolution structures of mitochondrial glutaminase C tetramers indicate conformational changes upon phosphate binding JOURNAL OF BIOLOGICAL CHEMISTRY Nguyen, T. T., Ramachandran, S., Hill, M. J., Cerione, R. A. 2022; 298 (2): 101564

  Abstract

  The mitochondrial enzyme glutaminase C (GAC) is upregulated in many cancer cells to catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. The dependence of cancer cells on this transformed metabolic pathway highlights GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescent reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the Y466W GAC tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric Y466W GAC bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e., the "lid"), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates, resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the Y466W GAC tetramer exhibited bound glutamine with closed lids. Glutamine can bind with high affinity to each subunit, which subsequently undergo simultaneous catalysis. These findings explain how the regulated transitioning of GAC between different conformational states ensures that maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.

  View details for DOI 10.1016/j.jbc.2022.101564

  View details for Web of Science ID 000761371500006

  View details for PubMedID 34999118

  View details for PubMedCentralID PMC8800119

• New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography JOURNAL OF BIOLOGICAL CHEMISTRY Milano, S. K., Huang, Q., Nguyen, T. T., Ramachandran, S., Finke, A., Kriksunov, I., Schuller, D. J., Szebenyi, D., Arenholz, E., McDermott, L. A., Sukumar, N., Cerione, R. A., Katt, W. P. 2022; 298 (2): 101535

  Abstract

  Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

  View details for DOI 10.1016/j.jbc.2021.101535

  View details for Web of Science ID 000761412100003

  View details for PubMedID 34954143

  View details for PubMedCentralID PMC8784640