Cells are tightly regulated environments where proteins need to be in exact the right place for proper function. Diseases such as cancers and neurodegenerative disorders often involve proteins being mislocated. In some cancers, for example, proteins that should monitor DNA replication in the nucleus are instead misplaced, allowing the cancer to progress.

Steven Banik, an assistant professor of chemistry at Stanford University and an institute scholar at Sarafan ChEM-H, has developed a method to return these misplaced proteins to their proper locations within cells. His team created a new class of molecules, called "targeted relocalization activating molecules" or TRAMs, which use the cell’s natural protein shuttles to redirect proteins to the correct place. The research, published in Nature on September 18, offers a potential therapeutic strategy for addressing protein misplacement in various diseases and for creating new cellular functions.

“We are taking proteins that are lost and bringing them back home,” said Banik.

Shuttles and Proteins

Cells are divided into compartments like the nucleus, which houses DNA, and the mitochondria, the cell’s energy producer. Proteins, which drive numerous cellular processes, must be in the right location to function properly.

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

In some diseases, proteins are mutated and end up in the wrong location. This can render the protein ineffective or, in some cases, harmful. For example, in ALS, the FUS protein is sent from the nucleus to the cytoplasm, where it forms toxic clumps that ultimately kill the cell.

Banik’s team sought to counteract this by utilizing natural protein shuttles to transport misplaced proteins back to their correct location. However, the shuttles have their own functions, so they needed a way to convince the shuttles to carry additional cargo.

To achieve this, the team developed TRAMs—two-headed molecules where one head binds to the shuttle, and the other attaches to the misplaced protein, or passenger, allowing the shuttle to transport the protein to the right location.

Ready, Set, Test

The team focused on two types of shuttles: one that moves proteins into the nucleus and another that exports them. Christine Ng, a graduate student and first author on the paper, designed and built TRAMs to hitch passengers to these shuttles. If proteins originally in the cytoplasm ended up in the nucleus, it would indicate the TRAMs worked.

The first challenge was measuring the amount of protein in specific locations. Ng developed a new method to quantify this within individual cells, combining her chemistry background with new skills in microscopy and computational analysis.

“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.”

Her experiments showed that TRAMs could move proteins into and out of the nucleus. Through these tests, Ng established basic design principles, such as how strong a shuttle must be to transport its cargo.

The next step was to see if TRAMs could reverse disease-causing protein misplacements. The team successfully developed a TRAM that returned the FUS protein to the nucleus in cells from ALS patients. This reduced the toxic clumping of the protein and improved cell survival.

They also tested their TRAMs on a well-studied mouse mutation that makes the animals more resistant to neurodegeneration. This mutation sends a protein away from the nucleus, down the axon. Banik’s team engineered a TRAM to mimic this mutation, successfully transporting the protein down the axon and making the cells more resistant to neurodegenerative stress.

The main challenge the team faced was designing TRAMs that can specifically target passenger proteins, as scientists haven’t yet identified all the molecules that bind to these passengers. To overcome this, they used genetic tools to add a sticky tag to the proteins they wanted to move. In the future, they hope to find natural sticky tags on proteins and further develop TRAMs into therapeutic molecules.

While their current work focused on nuclear shuttles, Banik’s method could be applied to other shuttles, such as those that move proteins to the cell surface, where cells communicate with one another.

Beyond correcting protein misplacement, Banik also hopes to use TRAMs to introduce new functions into cells by sending proteins to areas they don’t usually access.

“It’s exciting because we are just starting to learn the rules,” Banik said. “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?”