The inside of a living cell is a tangled web of interconnected pathways, where different kinds of data are ferried by millions of proteins. Decrypting a specific sequence of events in this chaotic mess is hard. Stopping or tweaking that sequence without touching any of the other pathways is even harder.
But scientists studying a vital signalling network have, for the first time, found a way to pinpoint and shut down a single chain reaction driven by a key protein without affecting the protein’s other tasks. Specifically, they used artificial antibodies that bind to the protein and cut off a single pathway. The technique could be a powerful tool to precisely probe or rewire specific cellular events, according to its inventors.
Their results were reported in the journal Nature Nanotechnology on October 2, 2017.
“I think it is a very elegant study… a technical advance with interesting implications,” says Sandhya Visweswariah, a professor at the Indian Institute of Science, Bengaluru, who was not involved in the research. “I can see the utilisation in being able to understand biology better; maybe there will be more insight into the way pathways work, get turned on, etc.”
The network the researchers studied controls body functions such as sight, blood circulation and immune response, and is found in cells throughout the body. It is driven by proteins called G-protein coupled receptors (GPCRs). The hope is that the technique could help drug-development in the long term.
A majority of the cell’s activity is controlled by signals from GPCRs, which nest in the cell membrane and act as gatekeepers. They span the width of the membrane, with a docking spot outside and a tail that hangs inside the cell. When a ‘messenger’ molecule binds to the outer dock, GPCRs trigger changes in proteins that are attached to its tail (like a relay), which in turn affects another protein inside the cell and so on. Depending on what messenger binds to it on the outside, the ultimate result of this chain reaction is that the cell grows, multiplies or dies.
This relay is found in practically every organ, pulling the strings behind the body’s flight-or-fight response, the tightening of blood vessels or the rallying of defence mechanisms. The pharmaceuticals industry is keenly interested in tweaking their activity: about half of the currently prescribed drugs in the market act by targeting GPCRs.
One of the key players in the GPCR network is a protein called β-arrestin. β-arrestin keeps GPCRs in check by either binding to and cutting off their signalling or pulling them into the cell for digestion. Like many proteins that wear multiple hats, β-arrestin also drives other unrelated chain reactions, such as those involved in cell movement and programmed cell death. Interrupting just one or the other of its relationships with GPCRs without affecting its other tasks has proved challenging thus far, and is a widely recognised problem.
In this study, the researchers found that certain antibody fragments generated in the lab, called Fabs, were able to bind to β-arrestin and keep it from pulling GPCRs into the cell – while other actions went on uninterrupted. “Now we have a handle on selectively taking out one function while maintaining the other, and then we can study what happens to the cell, to the animal and so on,” says senior author Arun Shukla, an assistant professor at IIT Kanpur.
There are real-life scenarios where shutting down only one of β-arrestin’s functions could help. In patients with renal diabetes insipidus, for example, genetic defects cause β-arrestin to keep pulling GPCRs unnecessarily into the cell, ultimately upsetting the kidney’s water balance. Turning off just this action could prove useful in treating this condition.
To get antibody fragments that bind only to β-arrestin, Shukla’s team employed a technique that takes the genetic sequence of a fragment and fuses it with the DNA of viruses called bacteriophages. When the virus makes its coat protein, it will also make the fragment and literally display it on the outside of its body. In this way, Shukla & co. created a ‘library’ with millions of viruses each displaying an antibody fragment. The whole library was then exposed to β-arrestin, and those that stuck to it were selected.
The researchers tested their approach on several GPCRs, such as those that control blood pressure, the flight-or-flight response and pain. They believe that it can be applied to most other GPCRs in the body as well.
Shukla suggests that their technique could also offer an edge over existing ones such as the popular gene editing tool CRISPR/Cas9.
CRISPR/Cas9 might, for example, bring about an effect similar to what the antibody does by introducing a mutation in GPCR. “However, what you are doing then is modifying the receptor all the time in the cell,” says Visweswariah. “Here, if he has a way to control when the antibody is made by the cell, he can turn off the signal when he wants to” – the sort of control not possible with CRISPR/Cas9.
Shukla’s team has been experimenting with lab-made antibodies that latch on to β-arrestin and GPCRs so that they can study them more closely. Recently, they were looking at how β-arrestin coupled with a protein called clathrin, which kicks off the reaction that pulls GPCRs into a cell. Shukla and his team were trying to get antibodies that lock GPCR, β-arrestin and clathrin into a tighter complex to take a closer look at how they interacted.
Instead, they stumbled onto antibodies that did the opposite: disrupt the complex by cutting off the β-arrestin-clathrin cross-talk – while leaving the β-arrestin-GPCR link untouched. “It was a bit of a serendipitous discovery,” says Shukla.
Artificial antibodies are not new – but this is the first time that they have been used to shut down specific signaling pathways, according to Amitabha Chattopadhyay, a professor and GPCRs researcher at the Centre for Cellular and Molecular Biology, Hyderabad. He was not involved with the study.
Chattopadhyay points out several challenges to developing a generic tool that can fine-tune GPCR activity. For one, scientists don’t entirely know what many of the 800 different types of GPCRs look like, let alone how they function. For another, their networks resemble complex electrical circuits with signals flowing in all directions. “The major experimental challenge would be to judiciously design and synthesise the antibody fragment so that there is no cross-talk with other pathways,” he says. “Unfortunately, we don’t know all the pathways yet.”
Shukla’s approach sidesteps some of these issues by targeting a well-known process that is believed to be common to most GPCRs, no matter what they look like. It could also be applied to other signalling systems. “If you have a protein similar to β-arrestin, then our conceptual framework can be applied,” says Shukla. “If you can generate antibodies against that particular protein, which our technology can do, you can play with that signalling system as well.”