Blog post by Dr. Tamara Maiuri
Regulation of PAR levels in HD cells
For a little more mechanistic insight, we turned to the major regulators of PAR levels: PARP, which generates PAR, and PARG, which breaks PAR down. To get an idea of how well these two proteins are working in HD cells, I performed dose response curves with inhibitors against each.
Interestingly, despite higher overall PAR levels in HD cells, it takes less PARP inhibitor to reduce the response by half (HD cells have a lower IC50). This suggests that the PARP in HD cells isn’t working as well as it does in normal cells. Seems at odds with the elevated PAR levels, but it could be that there’s so much DNA damage in HD cells, the overall PAR is high even without PARP functioning at its max. These results and the experimental details have been deposited to Zenodo.
As for the PARG inhibition experiments, I didn’t use a large enough range of concentrations to get an accurate reading of the IC50. The results (or lack thereof) were nonetheless deposited to Zenodo. I’m currently repeating the dose-response experiment with a wider range of doses.
More huntingtin PAR-binding experiments
We previously confirmed one region of the huntingtin protein that binds PAR, which we named PBM3. This is a small discovery, but I always find it exciting to uncover something about how nature works. Even more fun is thinking of clever ways to use nature’s tricks to find out more about how nature works! We have some ideas coming down the pipeline for how to use PBM3 as a tool.
The first idea was to use PBM3 to measure the on-off rate of huntingtin PAR binding. Then we could compare normal versus mutant huntingtin to see if mutant huntingtin holds on to PAR more or less tightly than it should. Spoiler alert: this idea didn’t work.
The first problem was a high degree of variability in PAR binding between technical replicates. The PAR overlay by dot-blotting is good for a “yes or no” answer whether something binds PAR, but it is not quantitative. So I tried improving the reproducibility with slot blotting instead of dot blotting (slot blotting optimization experiments in this Zenodo entry.
The second problem was that the control peptide (a PBM3 mutant that cannot bind PAR), had a non-specific effect in the assay. Not sure how to interpret this, but for now, the only conclusion is that this method is not working (sad face). Suggestions are welcome to improve this protocol (deposited to Zenodo)!
Work is also in progress on getting to the bottom of the reproducibility issues between preps of full-length huntingtin: https://zenodo.org/record/3552780#.XdwBjZJKh-U
This work is funded by the HDSA Berman/Topper HD Career Development Fellowship.