Testing oxidative stress conditions for huntingtin-DNA repair protein interactions

Blog post by Dr. Tamara Maiuri

In the first blog post reporting on the Oxidative Stress Interactome Project, funded by the HDSA Berman/Topper HD Career Advancement Fellowship, I promised to pull huntingtin interactors out of patient-derived skin cells. And I will! These are a much better model than the “easier-to-work-with” HEK293 cell model. But a few more optimization steps are necessary. In the process of working out the fractionation and crosslinking conditions, I ran into some irreproducibility with the amount of DNA repair proteins coming down with huntingtin under oxidizing conditions. There are a few DNA repair proteins, such as APE1 and XRCC1, that we already know interact with huntingtin upon DNA oxidative damage. I use these as a gauge for whether the experimental conditions are right. Once everything is in place, we’ll get to the purpose of this project: to identify more of these oxidation-dependent huntingtin interactors in the hopes of targeting them for HD therapy.

These DNA repair proteins interact with huntingtin under conditions of oxidative stress. So maybe the irreproducibility problem comes down to the source of oxidative stress: it’s not consistent enough from experiment to experiment. I’ve been using potassium bromate, but it loses its potency rather quickly, has to be prepared fresh each time, and the stock has to be replaced often—not the best situation for consistency and reproducibility.

In this oxidative stress optimization experiment (posted on Zenodo), I tried hydrogen peroxide instead of potassium bromate. It’s very difficult to get hydrogen peroxide shipped to the lab, and we’ve been waiting for it to clear customs since January! So I used the old stock. Not too surprisingly, it wasn’t very good at inducing DNA damage-related huntingtin protein interactions. But I decided to post about this experiment because I learned something else: the fractionation method I’ve been using releases proteins from the nucleus, where DNA is stored, in the first step. And the huntingtin interactors are enriched in this fraction. This simplifies the fractionation process even further, because I can omit the second step from now on.

Next on the list of oxidative stress agents is 3-nitropropionic acid (or 3NP). This is a good one to try because it mimics what is happening in HD neurons by damaging mitochondria. In fact, mice treated with 3NP end up with degeneration in the same brain regions as HD patients, and show similar symptoms. 3NP worked quite nicely to induce huntingtin interaction with our “positive control” DNA repair proteins, APE1 and XRCC1 (check out the results on Zenodo).

Skin cells from patients are one of the best cell-based models we have to study HD. But they grow very slowly and don’t yield much protein to work with. So we have to wait for them to grow. In the coming weeks I will have enough cells to test the experimental system I’ve worked out in HEK293 cells. Let’s hope for a smooth transition!

 

 

4 thoughts on “Testing oxidative stress conditions for huntingtin-DNA repair protein interactions

  1. Hello there, I am a medicinal chemist just getting into HD area. Fascinated about your work…curious, do people use iron (3) as oxidant?

  2. We use a variety of oxidants: hydrogen peroxide, nitropropionic acid, potassium bromate. H2O2 is fine in vitro, but not appropriate for live cells. KBrO3 can specifically oxidize DNA/RNA. NPA poisons mitochondria to mimic ROS stress from mitochondrial dysfunction.
    In studies where huntingtin is knocked down in adult mice, they see significant defects in iron homeostasis, likely related to huntingtin functions at the recycling endosome defined by Frederic Saudou. We think huntingtin function in endocytosis is about a ROS stress response to try and turn over iron III to reduce ROS.

    Elimination of huntingtin in the adult mouse leads to progressive behavioral deficits, bilateral thalamic calcification, and altered brain iron homeostasis.
    http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006846

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