News Release 

Synthetic llama antibodies rescue doomed proteins inside cells

Columbia University Irving Medical Center

Research News

Columbia researchers have created a new technology using synthetic llama antibodies to prevent specific proteins from being destroyed inside cells. The approach could be used to treat dozens of diseases, including cystic fibrosis, that arise from the destruction of imperfect but still perfectly functional proteins.

In many genetic diseases, including cystic fibrosis, mutated proteins are capable of performing their jobs but are tagged for destruction by the cell's quality control mechanisms.

"The situation is analogous to ugly fruit," says Henry Colecraft, PhD, the John C. Dalton Professor of Physiology & Cellular Biophysics, who led the research. "Shoppers reject fruit that doesn't look perfect, even though ugly fruit is just as nutritious. If mutated proteins in cystic fibrosis can escape the cell's quality control mechanisms, they work pretty well."

In the cell, proteins destined for destruction are marked with a small peptide called ubiquitin. Deubiquitinase enzymes (DUBs) can remove these tags, but simply increasing DUB activity would indiscriminately rescue all proteins in a cell marked for destruction, which would be harmful.

"A lot of proteins are destroyed by the cell for good reason," Colecraft says, "so a therapy needs to be selective."

That's when Colecraft and his graduate student, Scott Kanner, realized they could develop a solution that takes advantage of nanobodies--small antibodies produced naturally by llamas, camels, and alpacas that were discovered nearly 30 years ago. These small nanobodies bind their targets with exquisite specificity and retain this property inside cells, unlike regular antibodies.  

The new technology--called engineered deubiquitinases or enDUBs for short--combines a synthetic nanobody that recognizes a specific protein with an enzyme that can rescue proteins tagged for destruction.

In a new paper in Nature Methods, the researchers tested two different enDUBs, one designed to rescue a protein mutated in cystic fibrosis and another designed to rescue a protein mutated in long QT syndrome, an inherited heart disease that can cause arrhythmia and sudden death. 

To build each enDUB, the researchers first had to find a nanobody that only recognizes and binds the target protein. Until recently, researchers had to inject their target proteins into llamas, camels, or alpacas and wait for the animal to generate such nanobodies. The Columbia researchers instead fished out binders from a synthetic yeast nanobody display library containing millions of unique nanobodies.

Once created, each enDUB was tested in cells that produced the mutated proteins. 

In both cases, enDUBs prevented the destruction of the proteins, and the proteins migrated to their normal locations in the cell membrane where they performed their normal functions.

"In the case of one of the cystic fibrosis proteins we tested, we get a remarkable rescue, restoring protein levels in the cell membrane to about 50% of normal," Colecraft says. "If that happened in a patient, it would be transformative."

Though both diseases investigated in the study are caused by mutations in ion channel proteins, "the approach can be applied to any protein in the cell, not just membrane proteins or proteins altered by genetic mutations," Colecraft says. 

"It could be applicable to any disease where protein degradation is a factor, including cancer and epilepsy."

More Information

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The paper, "Targeted deubiquitination rescues distinct trafficking-deficient ion channelopathies," was published Nov. 9 online ahead of print in Nature Methods.

Scott Kanner is a graduate student in the Doctoral Program in Neurobiology and Behavior at Columbia University Vagelos College of Physicians and Surgeons.

Other authors: Zunaira Shuja (Columbia), Papiya Choudhury (Columbia), and Ananya Jain (Columbia, now at IQVIA).

This work was supported by the U.S. National Institutes of Health (grants RO1-HL121253, RO1-HL122421, T32 GM007367, 1F30-HL140878, S10RR027050, P30 CA013696) and the TRx Accelerator of the Irving Institute for Clinical and Translational Research at Columbia University Irving Medical Center (supported by UL1TR001873).

Scott Kanner and Henry Colecraft have filed a patent application through Columbia University based on this work. Columbia Technology Ventures is helping to commercialize this technology.

Columbia University Irving Medical Center provides international leadership in basic, preclinical, and clinical research; medical and health sciences education; and patient care. The medical center trains future leaders and includes the dedicated work of many physicians, scientists, public health professionals, dentists, and nurses at the Vagelos College of Physicians and Surgeons, the Mailman School of Public Health, the College of Dental Medicine, the School of Nursing, the biomedical departments of the Graduate School of Arts and Sciences, and allied research centers and institutions. Columbia University Irving Medical Center is home to the largest medical research enterprise in New York City and State and one of the largest faculty medical practices in the Northeast. For more information, visit cuimc.columbia.edu or columbiadoctors.org.

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