Title

Investigating the HSF1-HSF2 Interaction

Document Type

Poster

Publication Date

11-10-2017

Disciplines

Medicine and Health Sciences

Abstract

The over accumulation of misfolded protein aggregates is a key contributor in neurodegeneration associated with diseases such as Alzheimer’s, Parkinson’s, Huntington’s disease, and Amyotrophic Lateral Sclerosis (ALS). In a cell, a class of proteins known as chaperones (heat shock proteins) manages protein folding. The protein Heat Shock Factor 1 (HSF1) is the main regulator of transcriptional activation of these HSPs. It’s partner, HSF2, has been shown to moderate HSF1- faciliated expression of HSPs, acting as a limited participant in transcriptional activation of the heat shock response. The experiment investigated the HSF1-HSF2 interaction by addressing if they indeed interact and if so where this interaction is occurring. The hypothesis was that these two proteins are interacting via their coiled coil domains, located on the leucine zipper 1-3. Streptactin beads were used to pull down HSF1 due to its affinity for the strep tag. Nickel beads have an affinity for the His tag so it was used to pull down HSF2. Protein purification was performed and both streptactin beads and nickel beads were applied consecutively, allowing only HSF1-HSF2 co-expressing heteromultimers to be pulled down. Gels and western blots were then run and it was shown HSF1 and HSF2 co-express and co-purify from E.coli lysate. Using in-fusion cloning, two bacterial strains were then generated. One strain expressed HSF1 lacking the coiled coil domain and the other expressed HSF2 lacking its coiled coil. Using an FPLC, the purification was run again and through immunoblotting and protein gels a significant decrease in HSF1-HSF2 interaction was observed. It was established that HSF1 and HSF2 directly interact and co-purify from E. coli lysate. It was also concluded that the deletion of the coiled coil domains results in a significant decrease in HSF1-HSF2 interaction.

Rights

Copyright 2017 all authors

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