A New Method to Correct Misplaced Proteins in Cells

Cells are highly controlled spaces that rely on every protein being in the right place. Many diseases, including cancers and neurodegenerative disorders, are associated with misplaced proteins. In some cancers, for instance, a protein that normally stands watch over DNA replicating in the nucleus is sent far from the DNA it is meant to monitor, allowing cancers to grow.

Steven Banik, assistant professor of chemistry in the School of Humanities and Sciences and institute scholar at Sarafan ChEM-H at Stanford University, and his lab have developed a new method to help force misplaced proteins back to their proper homes within cells. The method involves rewiring the activity of naturally occurring shuttles to help move proteins to different parts of the cell. The team has devised a new class of molecules called "targeted relocalization activating molecules" or TRAMs that convince these natural shuttles to take different cargo – like the proteins that get exported from the nucleus in some cancers – along for the ride. Published in Nature on Sept. 18, this strategy could lead to a therapeutic to correct the protein misplacement associated with diseases, and also to create new functions in cells.

We are taking proteins that are lost and bringing them back home."

Steven Banik, Assistant Professor, Chemistry, Stanford University

Shuttles and Passengers

Our cells contain many compartments, like the nucleus, the secure home of DNA, or the mitochondria, where energy is produced. In between all these compartments is the cytoplasm. All throughout the cell's many locations are proteins. They are responsible for all sorts of actions – building and breaking molecules, contracting muscles, sending signals – but for them to function properly, they have to do their respective actions in the right place.

"Cells are really crowded places," said Banik. "Proteins are whizzing through the crowd passing by all kinds of other molecules like RNA, lipids, other proteins. So a protein's function is limited by what it can do and by its proximity to other molecules."

Diseases will sometimes take advantage of this need for proximity by mutating proteins that might otherwise be able to protect a cell from damage. 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 makes the protein stop working altogether. Proteins that act on DNA, for instance, will not find any DNA in the cytoplasm and float off doing nothing. Other times, this movement leads to a protein becoming a bad actor. In ALS, for example, a mutation sends a certain 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 whether they could combat this purposeful misplacement of proteins by using other proteins as shuttles to carry passenger proteins to their proper home. But these shuttles often have other functions, so the team would need to convince the shuttle to take on cargo and transport it to a new place.

To do this, Banik and his team developed a new kind of two-headed molecule called a TRAM. One head is designed to stick to the shuttle, and the other is designed to stick to the passenger. If the shuttle is strong enough, it will carry the passenger to its rightful place.

Along for the Ride

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

The first challenge was immediate: there were no reliable methods to measure the amount of a protein in 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. A chemist by training, she had to learn new skills of microscopy and computational analysis to do this.

"Nature is inherently complex and interconnected, so it's crucial to have interdisciplinary approaches," said Ng. "Borrowing logic or tools from one field to address a problem in another field often results in very exciting 'what if' questions and discoveries."

Next, she put it to the test. Her TRAMs successfully moved passenger proteins into and out of the nucleus, depending on the shuttle they used. These early experiments helped her generate some basic "rules" for design, like how strong a shuttle had to be to overcome the passenger's tendency to pull in another direction.

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

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

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

In all these examples, the team faced an ongoing challenge: Designing the passenger-targeting head of the TRAM is difficult because scientists have not yet identified all the possible molecules that could bind to their target passengers. To get around this, the team used genetic tools to install a sticky tag onto these passengers. In the future, though, they hope that they will be able to find naturally occurring sticky pieces on these passengers, and develop TRAMs into new kinds of medicines.

Though they focused on two shuttles, the method is generalizable to any other shuttles, like those that push things to the cell surface, where communication with other cells occurs.

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 that they cannot normally access, creating new functions that we do not yet know are possible.

"It's exciting because we are just starting to learn the rules," said Banik. "If we shift 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 piece of biology could we understand?"