Blog post by Dr. Tamara Maiuri
One of the major findings of the HDSA-funded project to find huntingtin interactors relevant DNA repair was that many proteins that interact with huntingtin are modified by poly ADP ribose (PAR). Previous posts have described our hypothesis that huntingtin binds poly ADP ribose, our preliminary evidence that it does so, frustrating attempts to understand why it does so, and other confounding results.
I wish this post was about a major breakthrough clarifying everything… but it’s just another normal, incremental piece of the puzzle (aka: science).
The good news is that one of the potential PAR-binding motifs we identified by sequence analysis does in fact bind PAR as a peptide in an in vitro PAR overlay assay. Furthermore, if the critical arginine residues are mutated to alanines, then the peptide no longer binds. This data has been deposited to Zenodo.
The bad news is that initial, reproducible PAR binding results with full length huntingtin and an N-terminal fragment have become less reproducible with different preparations of huntingtin protein. A full analysis of which preps bind PAR and which do not, along with some preliminary results suggesting it could be due to a heparin purification step, can be found here. I’m continuing to work with Dr. Rachel Harding (huntingtin protein super-producer) to get to the bottom of it.
The jury is still out on the physiological function of huntingtin PAR binding. Some of our results suggest a role for PAR in huntingtin chromatin binding, but this is confounded by the apparent trapping effect of PARP inhibitors on huntingtin. Given the identification of a potential PAR binding motif within huntingtin (PBM3), I decided to try a chromatin retention assay with fragments of huntingtin containing this sequence.
Preliminary results are… you guessed it: confusing. Two fragments of huntingtin bearing the PBM3 sequence actually had reduced chromatin binding in response to oxidative stress. Experimental details and results have been deposited to Zenodo.
It’s too early to make hard conclusions, but the huntingtin 1208-1810 and 1775-2413 fragments may actually bind chromatin under untreated conditions and release upon KBrO3 treatment. This is difficult to interpret since endogenous full length huntingtin tends toward chromatin retention upon oxidative stress. If the potential PAR binding motif PBM3 plays any role in this behaviour, mutating it within these fragments will tell.
So that’s the plan.