Cells are highly controlled spaces that depend on every protein being in the right place. Many diseases, including cancers and neurodegenerative disorders, are associated with misplaced proteins. In some cancers, for example, a protein that normally oversees DNA replication in the nucleus is sent away from the DNA it is supposed to control, allowing cancers to grow.
Steven Banik, an assistant professor of chemistry in the School of Humanities and Sciences and a Sarafan ChEM-H Institute scholar at Stanford University, and his lab have developed a new method to help get misplaced proteins back to their proper homes inside 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 relocalization-activating molecules,” or TRAMs, which coax these natural shuttles to carry a different cargo (such as proteins that are exported from the nucleus in some cancers) on the journey. Posted on Nature On September 18, this strategy could lead to a therapy to correct disease-associated protein misplacement and also to create new functions in cells.
“We are recovering the proteins that have been lost and bringing them back home,” Banik explained.
Shuttles and passengers
Our cells contain many compartments, such as the nucleus, the safe home of DNA, or the mitochondria, where energy is produced. Between all these compartments is the cytoplasm. Proteins are found in every location of the cell. They are responsible for all kinds of actions: building and breaking down molecules, contracting muscles, sending signals… but for them to work properly, they have to perform their respective actions in the right place.
“Cells are very busy places,” Banik says. “Proteins are zipping through the crowd, passing by all kinds of molecules, including RNA, lipids and other proteins. So a protein’s function is limited by what it can do and by its proximity to other molecules.”
Sometimes diseases take advantage of this need for proximity by mutating proteins that might otherwise 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 causes the protein to stop working altogether. Proteins that act on DNA, for example, find no DNA in the cytoplasm and float away doing nothing. Other times, this movement causes a protein to become 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 if they could combat this intentional protein misplacement by using other proteins as shuttles to carry the passenger proteins back to their original location. But these shuttles typically have other functions, so the team would have to convince the shuttle to accept a cargo and transport it to a new location.
To accomplish this, Banik and his team developed a new type of two-headed molecule called a TRAM. One of the two-headed molecules is designed to attach to the shuttle, and the other to the passenger. If the shuttle is strong enough, it will carry the passenger to his or her rightful place.
Throughout the journey
The team focused on two types of promising shuttles: one that drags proteins into the nucleus and another that exports them from the nucleus. Christine Ng, a graduate student in chemistry and first author on the paper, designed and built TRAMs that link shuttle and passenger. If a passenger in the cytoplasm ended up in the nucleus, she would know her 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, she had to learn new skills in microscopy and computational analysis to accomplish this.
“Nature is inherently complex and interconnected, so it’s crucial to have interdisciplinary approaches,” Ng said. “Borrowing logic or tools from one field to tackle a problem in another field often leads to very interesting ‘what if’ questions and discoveries.”
She then put it to the test. Her TRAMs were able to shuttle passenger proteins in and out of the nucleus, depending on the shuttle they used. These early experiments helped her generate some basic “rules” for the design, such as how much strength a shuttle had to have 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, reversing the movement of disease-causing proteins. First, they created a TRAM that relocated FUS, the protein that is sent out of the nucleus and forms dangerous clumps in ALS patients. After treating cells with their TRAM, the team saw that FUS was transported back to its natural home in the nucleus, and that the toxic clumps diminished 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 move away from the nucleus and down the axon of neurons.
The team wondered if they could build a TRAM that mimicked the protective effect of the mutation, by bringing the protein all the way to the end of the axon. Their TRAM not only caused the target protein to move down the axon, but also made the cell more resilient to the stress that mimics neurodegeneration.
In all of these examples, the team faced a constant challenge: designing the TRAM head for passenger transport is difficult because scientists have not yet identified all of the possible molecules that could bind to the passengers it targets. To get around 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 naturally occurring sticky bits on these passengers and develop TRAMs to turn them into new types of drugs.
Although they focused on two shuttles, the method is generalizable to any other shuttle, such as those that push things to the surface of the cell, where communication with other cells occurs.
And beyond sending mutated proteins back where they belong, the team also hopes that TRAMs could be used to deliver healthy proteins to parts of the cell they can’t normally access, creating new functions we don’t yet know are possible.
“It’s exciting because we’re just beginning 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 might we discover? What new part of biology might we understand?”
Banik is also a member of Bio-X and the Wu Tsai Human Performance Alliance. Other Stanford co-authors include Aofei Liu, a former graduate student in chemistry, and Bianxiao Cui, the Job and Gertrud Tamaki Professor of Chemistry. Cui is a member of Bio-X, the Cardiovascular Institute, and the Wu Tsai Neurosciences Institute, and is a faculty member of Sarafan ChEM-H. This work was supported by an A*STAR fellowship and by the NIH/NIGMS.