Researchers at the Université de Montréal in Canada responsible for developing an increasingly used live-cell ß-arrestin recruitment assay four years ago have now devised a way to monitor ubiquitination in living cells in real time using the same molecular biology technique.
The research, published in the December issue of Nature Methods, marks a significant advance in basic cell biology research, and may serve to provide a simple way for drug developers to assay for the activity of ubiquitin, a protein involved in a wide variety of disease states including cancer, Down syndrome, cystic fibrosis, and several neurodegenerative disorders.
Drug researchers are just beginning to find the ubiquitin pathway to be an effective drug target, diverging from the “big three” target groups: ion channels, GPCRs, and kinases and phosphatases. Ubiquitin is a protein found in the cells of all living creatures, and is most known for its role in targeting proteins for degradation. Ubiquitin has also recently been implicated in cellular functions such as intracellular signaling, sub-cellular localization, and protein-protein interactions.
The charge towards ubiquitination-targeting drugs has been led by the early successes of Velcade, an anti-melanoma drug that arrests ubiquitination, and the first oncology drug marketed by Millennium Pharmaceuticals.
But according to Université de Montréal professor Michel Bouvier, corresponding author on the study, Velcade is far from a perfect drug.
“[Velcade] targets ubiquitin-mediated proteolysis, and it’s working for different melanomas,” Bouvier told Inside Bioassays. “One of the potential problems with such a drug is the lack of specificity, because you’re inhibiting the entire proteome degradation system, so it may not be good for healthy cells.”
According to Bouvier, many researchers are currently attempting to target a specific ligase called E3 that is responsible for the ubiquitination of specific proteins, which may result in a much more specific drug for certain cancers.
“Many companies right now are screening for E3 ligase inhibitors,” Bouvier said, adding that the new assay should be suitable for monitoring the ubiquitination of specific substrates — including E3 ligase-mediated ubiquitination — and testing compounds for their ability to inhibit this in live cells on microwell plates.
The key molecular biology technique is called bioluminescence resonance energy transfer (BRET), and is a variation on its popular cousin fluorescence resonance energy transfer (FRET). In FRET, researchers can monitor the interaction between two proteins by attaching fluorescent proteins such as GFP and YFP to each of them. A transfer in energy between the fluorescent proteins occurs when they are very near one another — like when the proteins in question are interacting.
FRET has become very popular for live-cell assays because it can be performed with GFP and variants such as YFP and CFP. But one of the problems associated with it is that it requires readout instruments that are equipped with a laser for fluorescent protein excitation. Furthermore, the excitation laser often unintentionally excites the accepting fluorescent protein, making for some complicated mathematical analysis to extract the true FRET signal.
BRET doesn’t suffer from such a limitation because the donating protein is the expressed protein luciferase, which requires chemical activation instead of laser excitation.
“Not only that, but because of the lifetime of the excited enzyme, compared with the lifetime of the excited fluorophores in FRET, your chances of having a transfer are higher, which means that you need to play less with the linkers when you generate your fusion proteins,” Bouvier said. “You need to play much less with the linkers that you’re using.”
Furthermore, he said, BRET may lend itself to more efficiently multiplexed assays, something that the researchers explored in the recently published research.
“We used two generations of BRET, and we’re working on a third and fourth in the lab,” Bouvier said. “The substrate will determine the wavelength of the luminescence emission. So depending on the wavelength of the luminescence emission, you can pick the partners that you’re going to use.
“In the same assay, in the same cells, you can put protein A with luciferase, protein B with GFP, and protein C with YFP, and then you can simultaneously look at interactions between the three partners,” he added.
PKI Reaps Benefit
PerkinElmer owns the intellectual property behind the BRET technique, which was developed several years ago in collaboration between Bouvier’s lab and a Montreal-based biotech company called BioSignal. That company was acquired by Packard BioScience, which was subsequently acquired by PerkinElmer.
It is not the first time BRET has been used in a cell-based assay. One of the most well-known examples is the work Bouvier alluded to that was published in PNAS in 2000 [2000 Mar 28; 97(7): 3684-9], which used the technique in an assay for ß-arrestin activity in live cells. That assay, according to Bouvier, has become popular as a general assay for GPCR activity.
“The use of this for the ubiquitin is something that’s completely new,” Bouvier said. “It was not known if we would be able to tag the ubiquitin with GFP and still get a good BRET signal with a substrate.”
To get an adequate signal, Bouvier said, the researchers “had to play a trick” by mutating the ubiquitin so it would not form chains — something it tends to do and would lead to difficulties in accurately interpreting the results. “We test that in the paper, showing that if we don’t mutate the ubiquitin, you get a signal, but you get a much smaller signal,” he said.
Even with the modification made in this case, BRET signals can be weak, leading to one of its major drawbacks compared with FRET — it is very difficult to obtain actual images. As drug-discovery shops steadily begin to adopt high-content screening and basic research moves toward the concept of systems biology, researchers want to know not only when intracellular events are happening, but where as well.
Bouvier said that his lab is already working on a solution to the image problem. “There are a couple of ways to improve it,” he said. “Better cameras is one, and better cameras are being commercialized all the time. The one we have right now is better than the one we had five years ago, and we’ve started seeing some nice things.
“But you can also improve it by increasing the luminescence intensity,” he added. “We’re working with some enzymes right now which are different, which have ten-fold higher yields in terms of light emission. We have not done BRET with that yet, because we need to find the right partners, and tinker around with the enzyme to make it happy in certain conditions. But we are hopeful that these enzymes will produce much more light — enough to be able to take images.”
So this makes BRET a double-edged sword. On one hand, true images are hard to come by. But on the other hand, much simpler instrumentation can be used to monitor the assay over time. In the Nature Methods paper, the researchers used a simple luminescence detection instrument from the former Packard BioScience for one set of BRET measurements. For weaker signals, they used a more sensitive instrument from Berthold Technologies in Germany, which Bouvier opined was “the most sensitive for luminescence detection” on the market. Bouvier also said that instruments from PerkinElmer, BMG Labtech, and Molecular Devices could be used.
The ubiquitination application is already covered under the general BRET patent, so for commercial purposes, a lab would need to sublicense the IP from PerkinElmer.
And although the paper is only about a week old, an undisclosed company in France has already contacted the lab about how it might use the technique in its drug discovery programs. Bouvier said that it’s early to tell how effective a screening tool BRET-based ubiquitination might be, but said that the lab “is already working on the next generation of the assay.”