Next steps in the identification of ROS-related huntingtin protein-protein interactions

Blog post by Dr. Tamara Maiuri

In my last real-time report of the HDSA-funded project to identify of oxidation-related huntingtin protein-protein interactions, I was happy to report the successful purification of huntingtin and its interacting proteins from mouse cells. I was quite optimistic that the experiment would work using cells from an HD patient. This turned out not to be the case. Despite growing large amounts of cells, there was simply not enough starting material. Although we want to answer our questions about HD using human sources of information, it is just not technically possible with patient fibroblasts.

The good news is that I was able to generate two more replicates of the experiment in mouse cells. The total list of proteins identified by mass spectrometry can be found on Zenodo, and further refinement of the data was done by quantifying the intensity of each peptide (bit of protein) to give us a better sense of the most abundant hits. This has also been deposited on Zenodo.

Sifting through the data is taking some time—being a scaffold, huntingtin interacts with several hundred proteins. We are also in the final revision stages of a few manuscripts for which experiments have been prioritized (one manuscript describes how we turned HD patient skin cells into a tool for the HD research community—a pre-print can be read on Bioarchive). I will post a more detailed analysis in the coming weeks, but here are some general conclusions from the most reproducible results:

Stable interactions:

The proteins that interact with huntingtin in cells treated with DNA damaging agents also interact with huntingtin in untreated cells. This could be because

• The treatment didn’t work, or the untreated cells are under an unintended form of stress
• Huntingtin transiently “samples” interactions with many proteins in unstressed conditions, which it binds more tightly upon stress. In this case, the cross-linking step may cause us to capture weak interactions
• Some of the interactions may be non-specific artifacts of the experimental set-up

These possibilities will be tested by following up on interesting hits in our human fibroblast system.

A connection to poly ADP ribose:

Many of the proteins that interact with huntingtin are also found in data sets of “PARylated” and “PAR-binding” proteins (see references below). Poly ADP ribose, or PAR, is a small biomolecule that plays a role in the process of DNA repair (among many other cellular processes). When the DNA repair protein “PARP1” notices some damaged DNA, it starts to attach chains of PAR to nearby proteins. This forms a sort of net to recruit other DNA repair factors. The overlap between our list of huntingtin interacting proteins and PARylated/PAR-binding proteins suggests that huntingtin may also bind PAR, just like many other DNA repair proteins. In fact, I have preliminary results suggesting it does just that. I will post them soon!

Data sets of PARylated and PAR-binding proteins:

Gagné J-P, Isabelle M, Lo KS, Bourassa S, Hendzel MJ, Dawson VL, et al. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res. 2008;36: 6959–6976.

Jungmichel S, Rosenthal F, Altmeyer M, Lukas J, Hottiger MO, Nielsen ML. Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses. Mol Cell. 2013;52: 272–285.

Zhang Y, Wang J, Ding M, Yu Y. Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome. Nat Methods. 2013;10: 981–984.