Blog post by Dr. Tamara Maiuri
Well it’s been a long haul, but I’m happy to say I finally have a list of proteins that interact with the huntingtin protein (expanded versus normal) under conditions of reactive oxygen species (ROS) stress. This is the very first step to achieving the goal of the project: to identify drug targets that are relevant to the process of DNA repair, which, through powerful genetic studies, has been repeatedly implicated in the progression of HD.
This first step was not without its obstacles. The goal at the outset was to identify proteins out of real HD patient cells, a more relevant system than cells from an HD mouse model. Unfortunately, it’s nearly impossible to grow up enough cells to yield the protein needed for mass spectrometry. My solution to this problem was to treat cells in batches, snap freeze them, and store them for processing once I had enough.
After working out the conditions for cross-linking and fractionation, inducing oxidative stress, and pulling huntingtin-associated proteins out of HD patient cells, I started growing up batches of cells. On the day I harvested the largest batch yet, the ROS inducer, 3NP, didn’t show the tell-tale signs of working (floating cells, larger cell pellet). When I tested a sample for interacting DNA repair proteins, I found almost no interaction. That was a bad day. This batch cannot be used–it amounted to a waste of time and resources. I spent a few weeks trying to figure out what went wrong with the 3NP, but no dice.
At this point, it was time to consider options and cut losses: we need to move on with this project. So, I considered switching to mouse striatal cells. They may not be as accurate a model as human fibroblasts, but we can get a list of huntingtin-interacting proteins from mouse cells and verify them in human cells. I revisited the other ROS sources tested in the past, and decided on H2O2 (see the optimization experiment on Zenodo).
It was much faster growing up enough striatal cells for mass spec analysis. The experiment wasn’t perfect–the untreated HD cells showed undue signs of stress, and so this will have to be repeated to be sure of our results. But we now have a list! Here are the preliminary results, in a nut shell:
After eliminating likely false positives (ribosomal proteins, chaperones, cytoskelton), there are:
- 92 proteins that interact with huntingtin under basal conditions and are released upon ROS stress
- 36 of these are inappropriately maintained by expanded huntingtin
- 38 new interactions formed upon ROS stress
- 29 of which do not happen with expanded huntingtin
- 52 proteins that interact with expanded, but not normal, huntingtin upon ROS stress
Of note, HMGB1 was identified in the immunoprecipitates from H2O2-treated cells (both Q7 and Q111), which we have previously identified as a huntingtin interacting protein. Further, the list of proteins from H2O2-treated Q111 cells includes FEN1, PCNA, Formin1 (the actin binding protein required for DNA repair), CENPJ, PARP8, and many other DNA repair proteins. The full list and experimental conditions are available on Zenodo.
The good news is that we now know these conditions worked very well in the mass spec analysis, and it may be feasible to grow up enough human cells after all. Since our TruHD-Q43Q17 cells (from a patient with 43 CAG repeats) grow the fastest, I started with those. Last week, I sent samples from the TruHD-Q43Q17 cells treated with a DNA damaging agent called MMS for mass spec analysis. It will take a few more weeks to get enough TruHD-Q21Q18 cells (from a spousal control). Stay tuned for the results!
This project is funded by the HDSA Berman/Topper HD Career Advancement Fellowship.