A genetically encoded toolset to decipher the biology of post-translational modifications in the mammalian proteome

Boston College, Chestnut Hill MA

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Abstract

Project Abstract Post-translational modifications (PTMs) dramatically expand the chemical and functional space accessible to proteins. Over the last 25 years, the landscape of known PTMs within the mammalian proteome has expanded at a dizzying speed, driven by the remarkable advances in mass-spectrometry based proteomics. However, functional consequences of most of these PTMs remain poorly characterized. At the core of this deep knowledge-gap – on a critically important facet of our biology – lies the difficulty of homogeneously generating eukaryotic proteins carrying a desired PTM at chosen site(s) to study how the presence of the modification alters the protein’s properties in vitro and in cellulo. For most PTMs, the precise biochemical origin is either poorly understood or challenging to reconstitute without additional pleiotropic consequences. The noncanonical amino acid (ncAA) mutagenesis technology, enabled by genetic code expansion (GCE), provides an exciting solution for this problem by enabling site-specific incorporation of a modified residue into virtually any site of any protein expressed in living cells. However, application of this technology in mammalian cells has faced significant technical limitations, including restricted structural diversity of ncAAs, inefficient incorporation, and difficulties in extending it to hard-to-transfect cells. Over the last five years, we have systematically addressed these challenges by: A) Establishing new platforms to incorporate new structural classes of ncAAs, B) Developing a mammalian cell-based directed evolution platform to engineer the ncAA-incorporation machinery, C) Creating optimized expression systems and delivery vectors for efficient ncAA incorporation in diverse mammalian cells and tissues. These advances have already enabled us to model several new PTMs in mammalian cells, including tyrosine sulfation and phosphorylation, arginine citrullination, dual acetylation/methylation of lysine, serotonylation of glutamine, etc. In the next five years, we will continue our quest to expand the scope of this technology for modeling an even larger subset of important PTMs associated with the mammalian proteome. Additionally, using our novel mammalian cell- based directed evolution platform, we’ll systematically optimize the ncAA incorporation machinery to improve its scope and robustness. Finally, using our powerful new ability to site-specifically introduce previously inaccessible PTMs into proteins expressed in mammalian cells, we will develop novel approaches to study several different facets of their biology: 1) Characterize the unique interactome of a PTM-labeled protein (using proximity labeling/MS-proteomics). 2) How PTMs alter the conformational dynamics of complex mammalian proteins (using single-molecule FRET). 3) How certain PTMs enhance the immunogenicity of human proteins (using novel display strategies). In addition, the research proposed here will fundamentally advance the scope of the GCE technology for application in higher eukaryotes for numerous additional applications, which will have broad and deep impact beyond the scope of this proposal.