A fresh approach to reassigning improperly positioned proteins within cells may pave the way for innovative treatments for cancer and neurodegenerative diseases.
Cells maintain a meticulously organized environment where each protein must reside in its designated area. Misplacement of proteins is linked to various diseases, including cancers and neurodegenerative conditions. For example, in some cancers, a protein that should monitor DNA replication in the nucleus is displaced, leading to uncontrolled cancer growth.
Steven Banik, an assistant professor of chemistry at Stanford University and a member of the Sarafan ChEM-H institute, along with his research team, has introduced a novel method to return these misplaced proteins to their correct locations within cells. Their technique involves modifying the use of natural protein shuttles, which help transport proteins to various cellular areas. The researchers have created a new category of compounds called “targeted relocalization activating molecules,” or TRAMs, that can influence these shuttles to carry proteins—like those expelled from the nucleus in certain cancers—where they are needed. This groundbreaking work, published in Nature on September 18, may lead to therapies to correct the issues caused by protein misplacement and even introduce new cellular functions.
“Our goal is to recover proteins that have gone astray and guide them back to their rightful place,” remarked Banik.
Shuttles and passengers
Cells comprise various compartments, such as the nucleus, which protects DNA, and the mitochondria, responsible for energy production. The cytoplasm lies between these compartments, housing numerous proteins engaged in diverse tasks—building and breaking down molecules, contracting muscles, and transmitting signals. For proteins to execute their functions efficiently, they must reside in the appropriate locations within the cell.
“Cells are incredibly crowded environments,” Banik noted. “Proteins navigate through the crowd, encountering a variety of other molecules like RNA, lipids, and other proteins. A protein’s effectiveness is limited by its location and its interactions with nearby molecules.”
Diseases can exploit this need for proximity by altering proteins that normally safeguard cells. Such mutations can be compared to mislabeling a package, sending proteins to incorrect destinations that would not occur in healthy cells.
Occasionally, this aberrant movement can render a protein nonfunctional. For example, proteins that interact with DNA may wander aimlessly in the cytoplasm without encountering any DNA. In other cases, improper relocation may convert a protein into a harmful entity, as seen in ALS, where a mutation results in a protein called FUS moving out of the nucleus into the cytoplasm, therefore aggregating into toxic clusters that ultimately lead to cell death.
Banik and his team contemplated whether they could address this deliberate misplacement by utilizing other proteins as shuttles to transport passenger proteins to their intended locations. However, since these shuttles typically have their own functions, it was essential to persuade them to carry additional cargo to a new destination.
To achieve this, Banik and his team created a two-headed molecule known as a TRAM. One head is crafted to attach to the shuttle, while the other is designed to connect with the passenger protein. If the shuttle exerts sufficient force, it will deliver the passenger to its proper location.
Along for the ride
The researchers concentrated on two types of shuttles: one that transports proteins into the nucleus and another that exports proteins from it. Christine Ng, a graduate student in chemistry and the lead author of the study, engineered TRAMs that successfully linked the shuttle to the passenger. If a passenger protein was found in the nucleus, it demonstrated that their TRAM had successfully worked.
The first hurdle was a lack of reliable techniques for measuring how much of a protein was located in a specific part of individual cells. Consequently, Ng developed a novel approach to quantify the amount and positioning of passenger proteins within a cell at a given moment. As a chemist, she had to acquire new skills in microscopy and computational techniques to carry out this task.
“Nature’s complexity and interconnectivity necessitate interdisciplinary strategies,” Ng explained. “By borrowing insights or tools from various fields, we can tackle problems in innovative ways, leading to exciting questions and discoveries.”
Subsequently, she put her design to the test. Her TRAMs effectively transported passenger proteins in and out of the nucleus, depending on the shuttle utilized. These preliminary experiments allowed her to formulate basic principles for design, such as determining the strength required from a shuttle to counteract the passenger’s tendency to move elsewhere.
The next challenge was to create TRAMs that could serve as potential medical treatments, reversing the movement of disease-causing proteins. The team initially designed a TRAM aimed at relocating FUS, the protein that is misdirected from the nucleus and forms harmful aggregates in ALS. After applying their TRAM to cells, they observed that FUS was retrieved back into the nucleus, leading to a reduction in toxic aggregates and decreased cell mortality.
Following that success, the team examined a well-studied mutation in mice known to enhance resistance to neurodegeneration. This mutation induces a specific protein to move away from the nucleus down the neuronal axon.
The team aimed to engineer a TRAM that mimics the protective benefits of this mutation, promoting the protein’s journey down the axon. Their TRAM not only successfully escorted the target protein along the axon but also fortified the cell against stress, simulating neurodegenerative conditions.
Despite their achievements, the ongoing challenge remained: Designing the passenger-targeting head of the TRAM is complex because many potential passenger molecules are still unknown. To overcome this, the team utilized genetic techniques to attach a sticky label onto the passenger proteins. In future research, they aspire to identify naturally occurring sticky components on these passengers and train TRAMs into novel therapeutic agents.
While the focus was initially on two types of shuttles, this method can apply to other shuttles as well, including those that transport substances to the cell surface for intercellular communication.
Beyond just redirecting mutated proteins, the team envisions TRAMs being used to deliver healthy proteins to regions within the cell that they cannot usually access, potentially revealing functions that are presently unknown.
“This is an exciting time, as we are beginning to discern the underlying principles,” Banik expressed. “If we alter the dynamics, enabling a protein to interact with new molecules in different cell locations at different times, what outcomes await? What new functions could we discover? What fresh insights into biology can emerge?”
Banik is also affiliated with Bio-X and the Wu Tsai Human Performance Alliance. Co-authors from Stanford include Aofei Liu, a former graduate student in chemistry, and Bianxiao Cui, the Job and Gertrud Tamaki Professor of Chemistry. Cui is involved with Bio-X, the Cardiovascular Institute, and the Wu Tsai Neurosciences Institute, and is a faculty fellow of Sarafan ChEM-H. The research received support from an A*STAR fellowship and the NIH/NIGMS.
