Bachelor of Arts
Stylianos P. Scordilis
HSP25, Mouse, Proteome, Co-immunoprecipitation, Mice, Heat shock proteins, Proteomics
The murine 25-kilodalton heat shock protein, HSP25, is a small, non-ATP dependent chaperone and actin binding protein that can be induced to varying degrees under different stress conditions, including exercise. While HSP25’s role as an actin-binding protein is well established and it is known to act as a general chaperone in vitro, it is unclear what client proteins HSP25 may act upon in the exercise-stressed sarcomere. Additionally, although HSP25 is known to interact with a variety of co-chaperones, its interaction with ATP-dependent heat shock proteins (which are necessary for the unbinding of substrate proteins) has only been established in vitro. In order to establish an “interactome” encompassing HSP25’s binding partners in the sarcomere, a co-immunoprecipitation assay was developed using anti-HSP25 antibody crosslinked to protein A/G magnetic beads. These beads were used to co-immunoprecipitate HSP25 and associated proteins from exercised mouse muscle lysate. Eluted proteins were identified using high pressure liquid chromatography-coupled mass spectrometry (HPLC-MS) and immunoblotting. This showed that HSP25 binds co-chaperones including small heat shock proteins and the ATP-dependent heat shock proteins HSP70 and HSP90. Client proteins were enriched for sarcomeric proteins and metabolic enzymes involved in ATP production.
Overall, this work presents a method for the co-immunoprecipitation of HSP25. It also suggests that HSP25 acts in concert with several small heat shock proteins as well as ATP-dependent heat shock proteins in order to facilitate the folding of sarcomeric proteins and metabolic enzymes in muscle.
Kolber, Natalie Sarah, "HSP25 interactome" (2017). Honors Project, Smith College, Northampton, MA.
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