Nanobody approach prevents culling of incomplete but still functioning proteins

To ensure the health of the cellular proteins, the ubiquitination system marks accidentally folded or damaged proteins as disruptive. However, the ubiquitination system can be too enthusiastic, targeting incomplete proteins, but still capable of performing its usual useful functions. Perhaps if you spare no incomplete proteins, they may serve therapeutic purposes.

This possibility is being investigated by Columbia University-based scientists. They are developing an artificial deubiquitin enzyme (enDUB) that can reverse the ruthless tactics of the ubiquitination system. Importantly, enDUB can distinguish between proteins that need to be saved and proteins that actually need to be destroyed.

Details of this work were recently published Nature method, In the article titled “”Targeted deubiquitination rescues a clear undertrafficking ion channelopathy.. This article uses a Colombian scientist’s approach that relies on new technologies incorporating synthetic llama antibodies to result in the destruction of incomplete but fully functional proteins, such as cystic fibrosis. Learn how to treat the disease.

“To rescue the functional expression of the heterologous mutant ion channels underlying long QT syndrome (LQT) and cystic fibrosis (CF), we have developed an enDUB that allows selective ubiquitin chain removal from target proteins. “I did,” wrote the author of the article. “In the LQT type 1 (LQT1) cardiomyocyte model, enDUB treatment normalized delayed rectified potassium currents and action potential duration. CF-targeted enDUB is a common and drug-resistant CF. Mutation, [drugs that have already been approved by the FDA].. “

enDUB is a modified deubiquitin enzyme. Like the normal deubiquitin enzyme, enDUB removes the ubiquitin tag (a small peptide that says “destroys me”) applied by the ubiquitination system. However, enDUB incorporates synthetic Nanobodies that recognize specific proteins.

Natural Nanobodies are small antibodies produced by llamas, camels and alpaca. Discovered nearly 30 years ago, these molecules, unlike regular antibodies, bind to the target with exquisite specificity and retain this property intracellularly.

Current research has produced Nanobodies that recognize and bind only selected targets. One of the targets was a CF mutated protein. The other was a mutated protein in LQT syndrome, a hereditary heart disease that can cause arrhythmias and sudden death.

“as a whole”, Nature medicine The article states, “EnDUB-mediated targeted deubiquitination is a powerful protein that not only corrects a variety of diseases caused by impaired ion channel transport, but also introduces new tools for in-situ ubiquitin coding. It provides a stabilization method. “

The Colombian team, led by Henry Colecraft, PhD, also said that deploying enDUB should be more effective than simply increasing DUB activity and indiscriminately rescuing all proteins in cells marked for destruction. Said. It is harmful to interrupt the destruction of all proteins in the cell.

Colecraft observed that in many genetic disorders, including cystic fibrosis, mutated proteins can perform their job, but are tagged for disruption by cellular quality control mechanisms. “The situation is like ugly fruit,” he said. “Even though ugly fruits are just as nutritious, shoppers reject fruits that don’t look perfect. If cystic fibrosis mutant proteins can escape the cell quality control mechanism, they will It works pretty well. “

“Many proteins are destroyed by cells for good reason,” Colecraft added. “So treatment needs to be selective.”

To build each enDUB, Colecraft and colleagues, including graduate student Scott Kanner, first had to find a suitable Nanobody. Until recently, researchers had to inject the target protein into llamas, camels, or alpaca and wait for animals to produce such Nanobodies. Instead, Colombian researchers have taken binders from a synthetic yeast Nanobody display library that contains millions of unique Nanobodies.

After creation, each enDUB was tested in cells that produced the mutant protein.

In both cases, enDUB prevented the protein from being destroyed, and the protein moved to its normal location on the cell membrane, where it performed its normal function.

“One of the cystic fibrosis proteins we tested offers the amazing relief of restoring protein levels in the cell membrane to about 50% of normal,” Colecraft reported. “If it happened to the patient, it would change.”

Both diseases investigated in this study are caused by mutations in ion channel proteins, but “this approach can be applied to any protein in the cell, not just membrane proteins and proteins altered by genetic mutations.” Colecraft continued. “It has the potential to be applied to any disease caused by proteolysis, such as cancer and epilepsy.”