Blog post by Dr. Tamara Maiuri
We’re going live! Welcome to the first of a series of blog posts aiming to report our findings in real time. This post reports the first steps in the Oxidative Stress Interactome Project, funded by the HDSA Berman/Topper HD Career Advancement Fellowship.
The Oxidative Stress Interactome Project
Oxidative stress is something that happens in our brains as we age, and the inability to deal with it properly has been linked to neurodegenerative diseases including HD as well as Alzheimer’s and Parkinson’s. We know that the huntingtin protein interacts with many other proteins in response to oxidative stress. These proteins come together to repair DNA damaged by oxidation. What if some of those protein-protein interactions have gone wrong when the huntingtin protein is expanded, as it is in HD? That’s what we aim to find out. Then we’ll look for drugs that might fix the problem.
Setting The Scene (aka optimizing experimental set-up)
I will be purifying the huntingtin protein, and any other proteins interacting with it, from skin cells that came from real HD patients. The skin cells are grown in a plastic dish. First, I’ll treat the cells with oxidants such as hydrogen peroxide or potassium bromate, to mimic the oxidation that’s happening in the aging brain. Then I’ll compare the list of “huntingtin interactors” in the presence or absence of oxidation, to see which ones are recruited to the job under conditions of oxidative stress. Down the road, I will compare the normal and expanded huntingtin protein. But one step at a time…
The interacting proteins will be identified by a technique called mass spectrometry. I’ll explain mass spec in a future blog post, because before we get to that step, several things need to be running smoothly. For example, there’s no sense sending your samples for mass spec analysis if not enough huntingtin (and its interactors) was purified from cells in the first place!
To set the scene, I tested two aspects of the experiment:
- How to break up the cells and get at the protein (fractionation)
- How to keep the proteins from coming apart during the purification process (crosslinking)
In a fractionation optimization experiment that you can read about on Zenodo, I tested sonication, which basically uses sound energy to break up the cell nucleus and release DNA-bound proteins. It turns out that although sonication released more proteins from the cells, it may have disrupted protein-protein interactions or interfered with the purification of huntingtin, since less huntingtin and its interaction partners were recovered in the end. However, I could see that many more proteins interact with huntingtin upon treatment of the cells with oxidants. Those are the ones we want to identify!
After further fractionation optimization, and comparing notes with other lab members who are purifying huntingtin from cells, I settled on treatment with DNase (a protein that chews up DNA) to release the DNA-bound proteins from the cell.
In a crosslinking optimization experiment (also on Zenodo), I tested 2 different ways to link proteins together. One of the crosslinking chemicals (Lomant’s reagent) was quite finicky and came out of solution. The other (paraformaldehyde, or PFA) actually seemed to improve the cell fractionation process. Both chemicals helped keep the interacting proteins together. Since PFA gave better results, this is the crosslinking agent I’ll move forward with.
The scene is coming together, but there are a few more things to do to make sure we get the best/most informative samples of huntingtin and its interactors. These optimization steps have been done in different types of human cells, because they grow quickly and yield good amounts of protein. Skin cells from patients are not as easy to work with, but it’s time to test the system in those “fibroblasts”. That’s what I’ll be doing next. Stay tuned!
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