Anti-Clumping Compound Could Help with Huntington's

Newswise — Experiments in test tubes and bacterial cells reveal that a small molecule that cells use to cope with water loss can keep proteins from clumping into the aggregates that characterize neurodegenerative diseases such as Huntington's and Alzheimer's, a University of Massachusetts Amherst scientist reports. The research sheds light on the pathology of such diseases and points the way to a potential therapeutic intervention. The findings also have applications in the biotech industry where protein clumping can complicate large-scale protein production.

UMass Amherst scientist Lila Gierasch and her collaborator Zoya Ignatova of the Max-Planck Institute of Biochemistry in Germany are co-authors on the study, which is published in the Aug. 7 issue of the Proceedings of the National Academy of Sciences.

Proteins are major players in the biological world—among other things, they make biochemical reactions go and serve as building blocks for much of the structural elements of organisms. But before any protein can fulfill its biological role, it must fold itself into its designated shape, or conformation. Incorrectly folded proteins don't work and diseases such as Parkinson's, Huntington's and mad cow are all associated with misfolded proteins that clump together.

While it is generally understood that proper protein folding happens spontaneously, scientists are still trying to understand what causes a particular protein to fold properly or go awry. The protein's local environment plays a role, and other molecules in the neighborhood can assist or hinder the process, says Gierasch.

"It's a delicate balance—proteins can tip to the dark side very readily," she says.

Recently researchers have developed techniques that allow them to follow the fate of individual proteins within cells. Using an experimental set-up that they designed to do just that, Gierasch and Ignatova decided to see how the small molecule proline influenced protein folding. Proline is often taken up by cells in response to water loss, and other molecules with similar cellular roles have been shown to inhibit improper folding.

"There have been conflicting results," says Gierasch, "In some cases these molecules inhibit misfolding, in some cases they promote it."

To investigate, Gierasch and Ignatova used a strain of E. coli that makes a protein with a fluorescent tag attached. When the protein unfolds from its proper conformation and starts to clump, its fluorescence increases and can be tracked in real time.

When the researchers treated the cells with salt—causing proline to accumulate inside—they found that the proline prevented the protein from misfolding into the insoluble aggregates that are associated with several diseases. Increasing proline inside the cells after clumping was underway did slow aggregation down, but couldn't stop it. Test tube experiments recapitulated these results.

Then the researchers fused the protein with a big chunk of Huntingtin, the protein associated with Huntington's disease, and again tracked proline's anti-aggregation abilities. They found that if a lot of proline was present early on, the protein was less likely to clump up. Proline didn't abolish clumping entirely however, if it was administered after the misfolding was already underway. But it did change the properties of the clumpy aggregates. They remained soluble in detergents, unlike the Huntington's disease amyloid aggregates, which aren't soluble in detergent.

A protein's naturally folded shape, or "native" state is dictated by many factors such as where it has positive and negative charges, which sections are water loving, whether it has sections that are acidic or alkaline, and where all these sections are in relation to each other. Gierasch suspects that the proline inhibits misfolding by interacting unfavorably with the protein's backbone and favorably with its folded surface, making the overall protein more compact. Even though proper folding is encouraged from an energetic point of view, once aggregation begins, it is hard to stop, she says.

"Proline may be preventing the formation of the little seeds that have to form to get aggregation going in the first place," she says.

The findings shed light on the pathology of several neurodegenerative diseases and suggest therapeutic interventions that deserve further exploration, says Gierasch. There's also a biotech application—those interested in mass producing proteins now may have a new anti-clumping compound in the toolbox.

"Nature has a way of coping with the misbehavior of proteins and has natural molecules for responding to stress," says Gierasch. "We took nature's way and deployed it in a model system where we know quite a bit about how protein aggregation happens."