Project Summary: Exploration of the HTT interactome

Five months ago, I made my first post outlining the goals of my project here at the SGC. I am writing again today to update you on the progress I have made toward those goals. As a brief overview, I was working on finding Huntingtin (HTT) interacting proteins, but why would this be important? If you’ve read any of my previous posts, you will know that a mutation in HTT causes Huntington’s Disease (HD), a progressive neurological disorder. You will also know that there are currently no therapies for this debilitating disease, in large part due to our lack of understanding of how HTT functions, or how this might be perturbed when HTT is mutated. A hugely impactful tool for understanding a protein’s function is to understand the structure. This is what initially kicked off the HTT project at the SGC, but since the project’s inception, there have been a few roadblocks. Due to the inherent nature of HTT, it is not easily imaged. HTT can be broken into 3 main domains, the C-terminal, N-terminal, and Bridge domains. The C-HEAT domain is quite mobile with respect to the other domains, which is very problematic for current imaging techniques which are hampered in their ability to resolve conformationally heterogeneous protein samples. Recently, another protein, called HAP40, was found to bind HTT. This protein seemed to lock HTT into a single conformation allowing for the first structure of HTT to be produced. Although this is a significant first step, this structure only represents about 75% of the protein. My goal this summer was to find proteins that can function similar to HAP40 in locking HTT into a single conformation, but for more localized structures that were missed in this structure mentioned above. To do this, the SGC compiled a list of possible interaction partners. This list was formed from previous databases from the CHDI, a literature review, and a BioID experiment. For the majority of the summer, I was devoted to producing as many of these constructs as I could. From a list of 118, 17 were produced, and 9 of those were produced at sufficient quantity and purity to be used for interaction experiments. More specific methods for the purification of these proteins can be found at this link: link.

The general procedure used to assess binding was based on a simple pull-down. The construct of HTT used during these sets of experiments was representative of mutant HTT with an extended polyQ length of 54. This protein was copurified with HAP40. Both HAP40 and HTT had tags helpful for purification, those being FLAG and His-Tags, respectively. By mixing the possible interaction partner with HTT and then pulling down HTT via the tags, we would expect to see the interaction partner to be pulled down as well if they are, in fact, interacting in a sufficiently tight manner to stay together during this process. Sadly, during the preliminary screening, I was able to do, there were no signs of interaction by this method. This, however, is not a conclusive result. So many factors affect interactions. It is possible that the movement of a tag or a change of buffer conditions could completely change the results we observed, so there is still lots of work to be done on this. It is very possible that HTT interacts with many proteins through weak and transient interactions which would be difficult to capture by many biophysical methods. For the screens that have been completed so far, there is a link here to the exact methods used: link . I, unfortunately, will not be continuing with these trials, but with a continued effort toward this project, I have hope that this will aid in our understanding of HTT.

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