BUFFALO, N.Y.—GTP is a nucleotide and a building block of RNA that plays a role in both RNA and DNA synthesis. It’s also known to feature in the development of cancer and other diseases, and GTP levels are noticeably higher in cancerous cells compared to regular cells. In hopes of better tracking this molecule in live cells and learning more about its effect on cancer, scientists from Roswell Park Cancer Institute—whose collaborators include scientists from the University of Texas Health Science Center at San Antonio—have engineered sensors capable of detecting GTP levels within living cells. The team’s findings, published in a paper titled “Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution,” appeared in Nature Methods in September.
“GTP plays an essential role in multiple cellular processes, including protein synthesis, cytoskeleton maintenance and function, nuclear and intracellular transport and intracellular signaling,” Dr. Anna Bianchi-Smiraglia, a postdoctoral researcher with the Department of Cell Stress Biology at Roswell Park and first author of the paper, said in a press release. “By inserting a yellow fluorescent protein into a particular bacterial protein and monitoring the comparative change in fluorescence that results when GTP binds to our sensors, we are able to detect and track changes in GTP levels.”
The genetically encoded sensors are known as GTP evaluators, or GEVALs. As noted in the Nature Methods paper, the sensors were developed “by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range.”
The sensors represent the first available technology for measuring GTP in live cells, as well as the first to track the spatio-temporal dynamics that affect GTP levels and activity.
“There are certain areas in the cell that have high GTP levels, and certain areas that have low [levels]. We do not understand why, but before our findings, there was no way to measure it,” reports Dr. Mikhail Nikiforov, professor of oncology in the Department of Cell Stress Biology at Roswell Park and senior author of the paper. “Many people thought of GTP like water—everywhere in the same amounts. Some said ‘no, maybe it’s not,’ but really, before the sensors came into play, there was no way to identify, to ask this question, whether GTP is higher in certain areas of the cell or linked to certain functions, or it is just something that is homogeneously distributed all over the cell. Our sensors show that it is not homogeneously distributed, it’s a gradient.”
“Our sensors represent a new and unique tool for assessing changes in GTP levels in cell populations and individual cells, which may in turn point the way to effective strategies for suppressing or even preventing tumor growth,” he added. “This is an early finding that will have to be further developed through additional research, but it suggests opportunities for developing therapies that interfere with GTP metabolism by targeting key enzymes—perhaps existing therapies as well as new drugs yet to be developed.”
Nikiforov tells DDNews that there are a number of avenues in which the team would like to advance this work, such as making them applicable to different types of cells and to target the sensors to specific parts of cells or organelles.
“We also would like to use this sensor as a surrogate for screening drugs or small-molecule inhibitors which would decrease the GTP levels in cancerous cells,” he says. “And some of the existing drugs do that, they act to decrease GTP in cancerous cells. Unfortunately those are very few, and there are no efficient means of identifying them. Our sensors, which directly match the GTP levels in the cell, we submit to facilitate this effort.”