Catching a Giant Protein: Five New Ways to Grab Huntingtin

Huntington’s disease (HD) is caused by a change in a single gene known as huntingtin. This gene contains the instructions for making the huntingtin proteinhuntingtin protein The protein produced by the HD gene.. Despite more than three decades passing since the HD gene was discovered, scientists are still trying to figure out exactly what the huntingtin proteinhuntingtin protein The protein produced by the HD gene. does in healthy cells, and how the HD version causes disease.

One reason progress has been so slow is that huntingtin is really hard to study. It’s huge, it’s folded in complicated ways, and the tools scientists had were not very good at grabbing it. In a new study, a team from Canada and Japan made brand-new chemical tools: tiny rings of protein called macrocycles that can grab huntingtin in five different spots. So what did they make, what did they learn, and why does it matter for HD?

A giant protein that’s hard to see

To understand the challenge, it helps to picture huntingtin. If most proteins are like ordinary household appliances with a single job, huntingtin is more like a full kitchen – built to support many different tasks at once.

It’s a very long chain that folds up into two big blobs connected by a bendy bridge. The genetic change that causes HD lives at the very beginning of the huntingtin proteinhuntingtin protein The protein produced by the HD gene., where a short repeating segment (made of a building block called glutamineglutamine the amino acid building block that is repeated too many times at the beginning of the mutant huntingtin protein) gets abnormally expanded. People usually have around 20–35 of these glutamines; in HD, that number climbs past 35 and can reach 50, 80, or more.

Huntingtin has a lot of jobs in the body. It helps move stuff around inside cells, enables cells to divide, and cleans up junk. But we don’t know exactly how it does all this.

We do know one thing for sure: huntingtin has a best friend called HAP40, a smaller protein that sticks to it, known as a binding partner. Until now, HAP40 has been the only binding partner for which scientists have a detailed structural picture. Pinning down what else huntingtin does, and with whom, has been limited by the lack of sharp, specific tools to grab it.

Building molecular grappling hooks 

To make better tools, the scientists used a clever system called RaPID. Think of RaPID as a huge speed-dating party between tiny peptidespeptides Small pieces of protein that signal in the body from one cell to another (little pieces of protein) and huntingtin. The team made a super-giant collection of about one trillion different tiny peptidespeptides Small pieces of protein that signal in the body from one cell to another — that’s 1 followed by 12 zeros!

Each peptide was bent into a closed ring, kind of like a rubber band. A looped or folded shape can grab onto things more easily than a straight strand, like a bent paperclip can hook onto objects, while a straight wire just slides past.

Every little peptide ring had its own name tag (made of DNA) attached to it. So when a ring was bound to huntingtin, the scientists could read its tag and know exactly which peptide it was. They did several rounds of “fishing” to expose the collection of ring peptidespeptides Small pieces of protein that signal in the body from one cell to another to huntingtin, either alone or bound to its friend HAP40. They narrowed down the pool to only the stickiest ones, and five ring peptidespeptides Small pieces of protein that signal in the body from one cell to another emerged as the best huntingtin binders: HL2, HL5, HD4, HHL1, and HHD3.

Seeing exactly where each peptide ring sticks

Finding peptide rings that stick to huntingtin is only the first step. Next, the scientists wanted to know where each one grabbed huntingtin, and how tightly. They used three cool methods to figure this out:

• Tests that measure how strongly the rings hold on. All five rings held on really tightly, strong enough to be useful tools, and maybe even a starting point for potential drugs that could fish out toxic huntingtin.
• A method called HDX-MSmultiple sclerosis a disease of the brain and spinal cord, in which episodes of inflammation cause damage. Unlike Huntington's disease, MS isn't genetically inherited. that shows which parts of a protein get covered up when a ring sticks to it. When a ring attaches to huntingtin, it shields certain spots, leaving a “footprint” that helps scientists figure out where the ring is landing.
• Cryo-electron microscopy (cryo-EM), which is a super-powerful microscope that freezes proteins and makes 3D pictures so sharp you can see single atoms. (Yes, it’s as cool as it sounds.) This showed the exact contacts each peptide makes with the huntingtin proteinhuntingtin protein The protein produced by the HD gene..

Here’s what they found: two rings (HL2 and HD4) grab huntingtin in spots far from its best friend HAP40.

Two other rings (HHL1 and HHD3) only work when HAP40 is there, squeezing into a little pocket right between the two friends and folding themselves into a shape like the number 8 to fit.

The last ring (HL5) only likes huntingtin when it’s alone, without HAP40.

And here’s an important point: the five rings grabbed healthy huntingtin and expanded huntingtin just as well, which is especially useful in a tool designed to study the protein in both healthy and sick cells.

Fishing for huntingtin’s friends in real cells

Useful tools need to work in real life, not just in test tubes. So the scientists added a little hook (called biotin) to their peptide rings, like a fishing hook on a line. Scientists used these peptidespeptides Small pieces of protein that signal in the body from one cell to another as bait to see if they could catch huntingtin in human cells grown in dishes, along with whatever else was sticking to it.

One friend of huntingtin stood out way above the rest – HAP40. It showed up over and over, in every cell type the scientists tried, including brain-like cells that are similar to the ones damaged in HD. Even in cells with longer CAG repeats (like Q45 or Q81), huntingtin always had HAP40 stuck to it.

Over 3,000 other proteins have been reported to touch huntingtin, but this experiment shows HAP40 is the one that’s really there all the time. This strongly suggests that huntingtin and HAP40 function as a single unit most of the time, and the “real” form of huntingtin inside cells may almost always be the huntingtin–HAP40 pair.

Why this matters for Huntington’s disease

These new rings aren’t medicines yet, and the scientists are careful not to say they are. But they open up lots of exciting doors.

First, they work just as well as the tools scientists used before to stick to and visualize huntingtin (called antibodies), but they’re smaller, cheaper, and can grab many different parts of huntingtin, not just one. They’re also small enough that they might be able to slip inside living cells and bind huntingtin, which current antibodies can’t do. That would let scientists study huntingtin inside living cells in ways that are currently impossible.

Second, the high-resolution 3D pictures of these peptide rings sticking to huntingtin are like blueprints for the design of future medicines. For example, the figure-8 shape shows two small pockets at the huntingtin–HAP40 meeting point that future medicines could aim at, potentially to “fish out” toxic huntingtin. The rings might also be used as the “grabbing” part of cool new tools like PROTACs (tools that tell the cell to throw huntingtin in the trash) or PET tracers that could help doctors visualize huntingtin inside the brains of patients.

Third, the fact that huntingtin is almost always with HAP40 inside cells reframes how we should approach it. If we want to understand what huntingtin does, or how to fix it when it changes and becomes toxic, we probably need to study it with HAP40, not by itself. Scientists now have a sharper set of tools to investigate the mystery of whether huntingtin can function without HAP40. 

Looking ahead

Like all science, this paper leaves plenty of questions. The rings grab healthy and HD huntingtin the same way, so they can’t yet tell us how to target only the harmful kind, which is something researchers are still working on.

The next steps will be testing them inside living animals, and redesigning them to sneak into cells. Some new possible binding partners of huntingtin were found too, like ACADVL and HEY1, for which more tests are needed to confirm.

This research gives the HD community a brand-new set of tools for a protein that has been really hard to grab. Just like amazing microscopes changed what scientists could see, these tiny peptide rings promise to transform what we can ask about huntingtin function, in health, in disease, and in the search for a cure.

Summary

• Huntington’s disease (HD) is caused by a change that makes the huntingtin proteinhuntingtin protein The protein produced by the HD gene. abnormally long. Scientists still don’t fully know what this protein does, because it’s huge and hard to study with existing tools.
• Using a system called RaPID, the team tested about one trillion tiny ring-shaped protein fragments and identified five that stick tightly and specifically to huntingtin, each at a different, well-defined spot.
• A high-resolution microscope showed exactly how the rings attach to huntingtin. Two of them even fold into a cool figure-8 shape to fit into a pocket between huntingtin and its best friend HAP40.
• When the rings were used to “fish” huntingtin out of real human cells, they almost always caught HAP40 along with it, no matter the cell type or the length of the CAG repeatCAG repeat The stretch of DNA at the beginning of the HD gene, which contains the sequence CAG repeated many times, and is abnormally long in people who will develop HD.
• These rings are relatively cheap, small, and flexible, so they could help create future HD medicines, brain-imaging tools, and new ways to destroy harmful huntingtin.