Steric-free labeling strategies to study disease-related non-histone substrates of post-translational modifications

Temple Univ Of The Commonwealth, Philadelphia PA

Investigators

Linked publications, trials & patents

Abstract

Principal Investigator/Program Director (Last, first, middle): Wang, Ross, E. Steric-free labeling strategies to study disease-related non-histone substrates of post- translational modifications The proposed research will utilize fluorine-based bioorthogonal reactions for steric-free labeling and chemical biology interrogation of disease-related post-translational modifications (PTMs) such as acetylation and O-linked N-acetylglucosamine. Despite recent advances in biomedical research, diseases are still haunting human beings. Many cancer types remain lethal, particularly at late stages, and are resistant to traditional therapy, while most inflammatory diseases result in chronic or long-term burdens, and there is a lack of selective immunosuppressives on the market. Thus, there is a need for new therapeutic approaches by exploiting novel proteins and protein-protein interactions (PPIs) as targets. Disease-related acetylation and O-GlcNAcylation have recently emerged as important biological pathways that could unravel such potential targets. These PTMs modify existing proteins with specific chemical functionalities to modulate protein function, and thereby mediate various cellular activities including activation, proliferation, and migration. Dysregulation of the related proteins has been reported to be key to certain human diseases such as cancer and inflammatory disorders. Yet, the identity of these non-histone proteins and PPIs has not been fully elucidated. Current research in this area is still at a preliminary stage, heavily relying on chemical proteomics, which directly tags target proteins within complex proteomes with alkyne or azide- modified pro-metabolites such as acetyl-CoA or glucosamine. These chemical tags, after their metabolic labeling onto protein substrates, can later be derivatized in situ through bio- orthogonal ‘click chemistry’ with a fluorophore for imaging or a biotin affinity probe for pull down and proteomics- based target identification. However, these alkyne/azide- based tags are bulky in length and size, and for many cases cannot be metabolized by PTM enzymes and were barely incorporated onto substrates, thereby limiting the related target identification. The proposed research will focus on developing an innovative chemical tagging approach which is steric free and can be broadly used for the global profiling of proteins and PPIs related to acetylation or O-GlcNAcylation. We hypothesize that unlike azides and alkynes, fluorine labeling can best mimic the intrinsic carbon-hydrogen bond and is thereby steric free and generally applicable to tag acetylation and O-GlcNAcylation. The first research thrust seeks to utilize the fluorine-displacement reaction toolkit to systematically interrogate acetylation- involved proteins and PPIs in breast cancer and T-cell activation. The second research thrust involves the new methodology development by combining the fluorine-displacement reaction toolkit with specific inhibitors of the acetyltransferase so that one can specifically label and profile potentially novel substrates and PPIs downstream of that isoform-specific acetylation enzyme. The fluorine displacement based chemical proteomics approach will also be tested on O-GlcNAc substrates to reveal novel PPIs related to prostate cancer. The final thrust will investigate new generations of fluorine-mediated chemical reactions for PTM labeling. Page 1