Scientists develop new method to relocate misplaced proteins

Cells are highly controlled spaces that rely on each protein being in the right place. Many diseases, including cancers and neurodegenerative disorders, are associated with misfolded proteins. In some cancers, for example, a protein that normally monitors DNA being copied in the nucleus is sent away from the DNA it’s meant to monitor, allowing cancers to grow.

Steven Banik, an assistant professor of chemistry in the School of Humanities and Sciences and an institute scholar at Sarafan ChEM-H at Stanford University, and his lab have developed a new method to help force misguided proteins back into their proper homes within the cells. The method involves rewiring the activity of natural shuttles to help move proteins to different parts of the cell. The team has devised a new class of molecules called “targeted translocation activating molecules,” or TRAMs, that coax these physical shuttles to carry different cargo—such as proteins exported from the nucleus in some cancers—along the way. Posted on Nature On September 18, this strategy could lead to a therapeutic correction of disease-related protein misplacement, as well as the creation of new functions in cells.

“We take proteins that are lost and bring them back home,” Banik said.

Buses and passengers

Our cells contain many compartments, such as the nucleus, the safe house of DNA, or the mitochondria, where energy is produced. Between all these compartments is the cytoplasm. All of the cell’s many sites are proteins. They are responsible for all kinds of actions – building and breaking molecules, contracting muscles, sending signals – but to function properly, they must do their respective actions in the right place.

Cells are really busy places. Proteins flow through the crowd passing through all kinds of other molecules like RNA, lipids, other proteins. So the function of a protein is limited by what it can do and by its proximity to other molecules.”

Steven Banik, assistant professor of chemistry in the School of Humanities and Sciences and Sarafan ChEM-H Institute Fellow at Stanford University

Sometimes diseases take advantage of this need for proximity by mutating proteins that might otherwise protect a cell from harm. These kinds of mutations are like putting the wrong address on a package, tricking proteins into going where they would never go in healthy cells.

Sometimes, this movement causes the protein to stop working altogether. Proteins that act on DNA, for example, will find no DNA in the cytoplasm and float around doing nothing. Other times, this move leads to a protein becoming a bad actor. In ALS, for example, a mutation sends a particular protein, called FUS, out of the nucleus and into the cytoplasm, where it aggregates into toxic clumps and eventually kills the cell.

Banik and his team wondered if they could combat this intentional protein misplacement by using other proteins as shuttles to ferry passenger proteins home. But these buses often have other functions, so the team will have to convince the bus to take the cargo and take it to a new place.

To do this, Banik and his team developed a new kind of two-headed molecule called TRAM. One head is designed to stick to the bus and the other to stick to the passenger. If the bus is strong enough, it will take the passenger to the seat he deserves.

Along for the ride

The team focused on two promising types of shuttles, one that drags proteins into the nucleus and another that extracts proteins from the nucleus. Christine Ng, a chemistry graduate student and first author on the paper, designed and built TRAMs that connect the bus and the passenger. If a passenger in the cytoplasm ended up in the nucleus, they would know that their TRAM had worked.

The first challenge was immediate: there were no reliable methods for measuring the amount of a protein at a specific location in individual cells. So Ng developed a new method to quantify the amount and location of passenger proteins within a cell at a given time. As a chemist by training, he had to learn new skills in microscopy and computational analysis to do this.

“Nature is inherently complex and interconnected, so it is critical to have interdisciplinary approaches,” Ng said. “Borrowing logic or tools from one domain to address a problem in another domain often leads to very exciting ‘what if’ questions and discoveries.

Then he put it to the test. Its TRAMs successfully carried passenger proteins in and out of the core, depending on which bus they were using. These early experiments helped her establish some basic “rules” for design, such as how strong a bus needed to be to overcome the passenger’s tendency to pull in the other direction.

The next challenge was whether they could design TRAMs that could be drugs, ones that reverse the movement of disease-causing proteins. First, they created a TRAM that would reset FUS, the protein sent out of the nucleus that forms dangerous granules in ALS patients. After treating the cells with their TRAM, the team saw that FUS was transported back to its natural home in the nucleus, and that the toxic clusters were reduced and the cells were less likely to die.

They then turned their attention to a known mutation in mice that makes them more resistant to neurodegeneration. The mutation, famously studied by the late Ben Barres and others, causes a particular protein to travel away from the nucleus down the neuron’s axon.

The team wondered if they could engineer a TRAM that would mimic the mutation’s protective effect by taking the protein for a ride to the end of the spindle. Their TRAM not only moved the target protein down the axis, but also made the cell more resistant to stress that mimics neurodegeneration.

In all of these examples, the team faced an ongoing challenge: Designing TRAM’s passenger-targeting warhead is difficult because scientists have not yet identified all the possible molecules that could bind to their target passengers. To overcome this, the team used genetic tools to install a sticky tag on these passengers. In the future, however, they hope to be able to find natural sticky bits in these passengers and develop the TRAMs into new kinds of drugs.

Although they focused on two shuttles, the method can be generalized to any other shuttles, such as those that push things to the surface of the cell, where communication with other cells takes place.

And beyond sending mutated proteins back to where they belong, the team also hopes that TRAMs could be used to send healthy proteins to parts of the cell they can’t normally access, creating new functions that don’t. we still know they are possible.

“It’s exciting because we’re just starting to learn the rules,” Banik said. “If we change the balance, if a protein suddenly has access to new molecules in a new part of the cell at a new time, what will it do? What functions could we unlock? What new part of biology could we understand?”