Decoding Membrane Proteins Crucial to Tissue Barriers

By Dirk Hoffman

The narrow spaces between cells represent natural weak points where viruses, bacteria, toxins, and other harmful substances can enter human systems.

Tight junctions are cell structures that form at these spaces and play a critical role by acting as barriers that block disease-causing agents,

Tight junctions are cell structures composed of membrane proteins that form within these spaces and play a critical role for tissues by acting as barriers that block disease-causing agents, according to Alex J. Vecchio, PhD, assistant professor of structural biology at the Jacobs School of Medicine and Biomedical Sciences.

“In humans, three families of membrane proteins in epithelial and endothelial tissues control the form and function of tight junctions. When these proteins assemble incorrectly, it can cause tissue-specific diseases through breakdown of tight junction barriers,” Vecchio says.

Because the structure of tight junctions is so complex, the research is akin to working on a 3D puzzle, Vecchio explains.

“The individual puzzle pieces, which are membrane proteins, are shaped uniquely to specifically interact with other proteins and form the larger 3D tight junction structure,” he says. “However, we don’t know what the shapes of these proteins are or what other proteins they interact with. So, it’s like trying to put a 3D puzzle together blindfolded and not knowing if you even have all the necessary pieces.”

Because of this, a model of the 3D structure of tight junctions cannot presently be devised.

“Our major goals are to determine what shapes tight junction proteins form and the rules that govern their interactions and assembly,” Vecchio says.

He said if the researchers know the shapes and interaction patterns of these membrane proteins, they can build more precise and complete models for how they fit together to solve the 3D puzzle.

“Solving the 3D structures of tight junctions will show us how they form barriers, which opens up a world of possibilities for therapeutic development,” Vecchio says.

Vecchio is principal investigator on a new National Institutes of Health grant that aims to uncover how tight junctions form naturally — and how their breakdown can lead to disease.

Titled “Structure and Assembly of Membrane Proteins at Tight Junctions,” the five-year, $2.1 million grant comes from the National Institute of General Medical Sciences. This work extends a Maximizing Investigators’ Research Award (MIRA) originally granted to Vecchio in 2020 when he was a faculty member at the University of Nebraska-Lincoln.

Vecchio explains that his group uses a multidisciplinary approach — combining structural biology and protein biophysics — to uncover how these membrane proteins assemble and regulate tissue homeostasis.

“Our approaches are novel because we view tight junctions through a structural biology lens. We think about how tight junctions form and function from the bottom up — from atoms to molecules to assemblies to cells to tissues — so from very small to very large. It’s the reverse of how some biomedical research is conducted,” he says.

“I think all these scales need to be strongly considered and integrated to understand tight junctions holistically.”

Vecchio emphasizes that this work requires expertise in biology, chemistry and physics and the merger of techniques associated with these disciplines to attain the levels of quantification and visual precision the research demands.

“For instance, immediately upon starting this work we had to figure out how to determine the shapes of tight junction membrane proteins more quickly and easily without sacrificing resolution,” he says. “These proteins are very small and dynamic, meaning they pose challenges for every major structural method.”

Vecchio says his group spent part of the last five years developing methods to enable structure determination of tight junction membrane proteins by cryogenic electron microscopy (cryo-EM).

Cryo-EM is an established technique but has benefitted from recent technological advances to revolutionize structural biology.

“In cryo-EM we shine electrons on proteins that are trapped in a thin layer of vitreous ice to cast their shadowed 2D projections. Because the proteins are trapped in random orientations, many 2D projections can be merged to form a complete 3D structure,” Vecchio says.

Cryo-EM works best to cast shadows of large and stable molecules, and so for the researchers to get it to work with small membrane proteins, they had to discover or design natural and non-natural molecules that bind tight junction proteins to make them larger and more stable.

“For this, we used antibody engineering platforms, toxins from bacteria, and synthetic proteins designed by artificial intelligence (AI). In the process, we found that many of these molecules alter the functions of cells and tissues, which is a thrilling and unanticipated result,” Vecchio says.

“Using this structural biology lens, we approach problems and ask questions from  distinct perspectives, which allows us to offer unique solutions and sometimes unexpected answers to the tight junction research community — and beyond,” he adds.

“Much of what we have and will continue to accomplish in this research stems from the flexibility provided by the MIRA grant mechanism. The freedom of inquiry it allows has facilitated our expansion into new techniques, the development of novel molecules, and establishment of collaborations across the U.S. and Europe.”

Because tight junctions are essential across all tissues, Vecchio says the direct clinical applications of his work are hard to predict. The research is focused on understanding the basic biology, rather than immediate therapeutic outcomes.

However, his group has already developed molecules that can open tight junctions —which could potentially be used to deliver drugs to normally restrictive tissues like the brain to treat central nervous system disorders.

The researchers have also discovered molecules that prevent a very prevalent form of food poisoning.

“Because tight junctions regulate passage of molecules between cells, we expect that our work will be used by others to aid the development of drug-like molecules that either facilitate or disrupt tight junction barrier function, depending on the application,” Vecchio says.

“The former can help treat diseases that result from barrier disruption, while the latter can empower delivery of drugs through tissues, which is currently a major challenge for central nervous system disorders.”

“Some of the drug-like molecules used in future therapies may be based on the molecules that we have developed and characterized, which is very motivating to me,” Vecchio says. “I am extremely excited to see what the next five years brings.”