A hallmark of cancers and neurodegenerative diseases is formation of stable amyloid protein aggregates. Ubiquitin is a small protein that, upon covalent attachment to lysiness, acts as a post-translational modification “tag” to condemn substrates to proteasomal degradation. Amyloid aggregates linked to pathologies are heavily ubiquitinated; however, it is unknown whether ubiquitination plays a role in inducing these misfolded states. It is possible that ubiquitin contributes to the misfolding process by destabilizing the fold of amyloid-forming proteins. However, such studies have been hampered by the need for large amounts of purified samples to study changes in protein stability, which is challenging for ubiquitinated proteins. Here, we develop a differential scanning fluorimetry (DSF)-based approach to measure the energetic effects of ubiquitin on the substrate protein to which it is attached. In this system, which we term “ortho-DSF,” we leverage a library of conformationally sensitive fluorogenic dyes to detect changes in stability upon ubiquitination for a model protein, barstar. In these experiments, Tm detected by a dye upon temperature denaturation is used as a proxy for protein stability. These dyes are “orthogonal” because they do not recognize ubiquitin. We have established a system using five ortho-DSF dyes to monitor the unfolding curves of barstar before and after ubiquitination at three sites. These sites are known to have different effects and, thus, serve as a proof of concept for using fluorogenic dyes to measure ubiquitinated protein stability. We expect that ortho-DSF will overcome the hurdles of traditional techniques that require large amounts of sample and specialized equipment, offering a unique avenue to study changes to protein energetics upon ubiquitination at scale. We plan to expand these studies to disease-associated amyloid-forming proteins to understand mechanisms of ubiquitin-influenced amyloid formation in disease.
Hopham et al. (Sun,) studied this question.