Researchers studying contact lenses discover new function for protein previously considered inert – ScienceDaily

The lens is composed of cells filled with structural proteins called crystalline. The crystals in each cell of the lens form a dense protein gel, and the optical properties of the gel, as well as its transparency and light refraction, help focus the light on the retina.

But when the crystalline proteins agglutinate, they are not so transparent anymore. If a sufficient amount of protein passes from their usual dense, water-soluble organization to dense aggregates, they begin to disperse the incoming light, forming cloudy deposits known as cataracts.

Eugene Serebryany, Harvard postdoctoral researcher, lead author of a recent study in the Journal of biological chemistry, researchers have long thought that crystalline proteins were chemically inert. That is, except for aggregating as an individual age, the proteins would not interact much with proteins of the same type. Serebryany said: "Such was the model: the actual function of (crystallin) is to remain monomeric and transparent and avoid aggregating it as long as possible".

At the time he was a student at MIT, Serebryany had used a mutant form of the gamma-crystalline lens protein to mimic the damage caused by UV rays to the protein. While studying how this mutation leads crystals to aggregate into aggregates, Serebryany found something amazing: the mutant was more likely to aggregate if a wild-type or undamaged protein was also present.

Harvard professor Eugene Shakhnovich, who collaborated with Serebryany and his graduate advisor, Jonathan King, described the discovery as "a pretty striking phenomenon" and explained, "If you had these damaged proteins in a test tube, they would not, if you had the wild-type protein, it would not aggregate forever, but then, when you mix the two, you see a quick and hasty aggregation. "

In other words, the healthy version of a protein that everyone thought was inert somehow caused a slightly damaged version to worsen – and quickly.

When Serebryany graduated, Shakhnovich hired him to continue working to understand how a supposedly inactive protein could cause this effect. Serebryany said, "The first thing to do was to try to make sure that the experiences of my PhD lab work in this (new) laboratory."

"They are only two subway stops!" Shakhnovich joked.

But, for some reason, Serebryany was struggling to replicate the results. "It's a different place, a different set of instruments, a slightly different set of procedures, you see where it's going," he said. "Suddenly, highly reproducible experiments before were very variable."

Indeed, in the Harvard lab, sometimes, the wild-type lens caused aggregation of mutant crystalline, and sometimes not. The scientists have been mystified.

Serebryany said: "Obviously, if there is sudden variability, there is a hidden variable that we had not seen before." He set up a series of experiments to identify this variable.

A close comparison of the molecular weights of the wild-type protein that caused the mutant to cluster together with the protein that did not reveal a difference equivalent to the weight of two hydrogen atoms. The researchers deduced that the state of redox – if two sulfur atoms in one molecule of protein were linked to each other instead of an atom Hydrogen – could make the difference.

"By performing isotopically resolved mass spectrometry experiments, we have achieved more than we expected," explained Serebryany. "Not only did the aggregation-prone mutant acquire an internal disulfide bond per molecule during the aggregation reaction, but the wild-type protein promoting aggregation lost its disulfide at the same time. "

By modifying the amino acid residues of cysteine ​​containing sulfur one by one into residues containing no sulfur, Serebryany discovered that two amino acids of cysteine ​​are close to one another at the surface gamma-d-crystalline acted as a sort of switch. When the two bound together, forming a structure called disulfide bond, crystallin appeared to be able to push the other damaged molecules toward aggregation. When the two cysteines were not linked, each took a hydrogen atom, explaining the minimal change in mass of the protein. Under these conditions, the wild type crystalline was inert.

But how could a link between amino acids on the surface of this protein cause other proteins to aggregate?

Using biophysical and biochemical techniques, the team found that although the disulfide bond is easily formed, it also introduces constraints into the structure of the protein. This made each molecule of protein capable of passing along the disulfide bond to a molecule close to the protein, in return receiving two protons. In this way, the disulfide bond could be constantly passed between the crystal protein molecules. The authors compared the process to a hot potato.

Given an entire population of healthy, undamaged crystalline proteins, this process could continue indefinitely. However, the authors showed that if a protein was already a little damaged, it caught the hot potato with another set of cysteines, which were less able to transmit it. This led the damaged protein to agglutinate. Previous work by the authors revealed that mutations mimicking the damage caused by UV rays changed the stability of the protein, making it more flexible, and therefore more likely to acquire the conformation that exposes new cysteines that can catch potato. hot.

This helps us understand the formation of cataracts. According to Shakhnovich, the team is working on peptide treatments that could prevent the "hot potato" from reaching the damaged proteins. Serebryany hopes that these peptides "could in fact absorb some of these disulfides and delay the time required for the formation of species most prone to aggregation". This may result in slower cataract formation in patients.

This study was funded by the National Institutes of Health.