Upgrading the mammalian translation system to probe multi-site post-translational modifications

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Proteins are molecular machines that drive the processes underlying biology. The function of proteins are frequently regulated by a wide-variety of reversible chemical modifications, such as acetylation of lysine residues. While numerous such acetylations have been identified on various human proteins, their role in biology remains unclear. Many human proteins are acetylated at multiple different sites, but how these individual modifications contribute to regulation remains poorly understood. The current inability to generate target proteins homogeneously acetylated at a set of chosen sites is a major challenge. This limitation will be overcome by establishing a novel technology. This technology will allow direct modification of a target protein during its synthesis in human cells. This will enable systematic elucidation of how multi-site acetylation regulates protein function. A cross-disciplinary summer research program will be developed to train and motivate high-school students from the greater Boston area. In the last two decades, rapid advances in analytical techniques have identified numerous human proteins that are acetylated at multiple sites. However, the daunting challenge of producing recombinant proteins in a homogeneous state of acetylation been a major roadblock. The genetic code expansion technology offers a powerful strategy to overcome this limitation, by allowing co-translational site-specific incorporation of N (epsilon)-acetyllysine (AcK) into recombinant proteins using an engineered nonsense suppressing aminoacyl-tRNA synthetase/tRNA pair. However, it is currently challenging to incorporate more than one AcK residue per protein in mammalian cells. To address this limitation, systematic engineering of the components of the mammalian translation system that limit the incorporation efficiency of AcK, will be carried out. A novel directed evolution platform for engineering these components based on their performance in live mammalian cells will be developed. The enhanced AcK-incorporation system will be used to interrogate how the function of HMGCS2 is regulated by the acetylation of three different lysine residues. Outreach efforts include a summer research program targeted to local high school students. This program called "You Evolve a Protein!(YEP!)" highlights the use of laboratory evolution to engineer proteins with desired properties. Starting from a fluorescent protein that emits green light, the participant will create engineered variants with altered color.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.