Our long-term goal is to understand the various regulatory roles of glycosylation. We are specifically interested in O-N-acetylglucosamine (O-GlcNAc) modification, where the GlcNAc moiety is attached by an ether linkage to a serine or threonine residue of a protein. This type of modification is ubiquitous in eukaryotic cells and may work in conjunction with phosphorylation to modulate the function of many proteins. However, the exact role of O-GlcNAcylation in the regulation of the structure and function of proteins had yet to be fully determined and exploited. One of the major bottlenecks for studying glycosylation is the inability to generate large amounts of homogenously glycosylated proteins. In this proposal, we would like to circumvent this problem by creating novel genetic codes and using them to `hijack' the translational machinery of the cell. Our system will be developed for mammalian cells, for this would allow the protein of interest to be produced in its native environment with necessary post-translational modifications, allowing for more natural folding, as well as the ability to perform functional assays in vivo. To generate novel genetic codes, we will evolve mutant tRNA-synthetase/suppressor-tRNA pairs that utilize glycosylated amino acids, and are orthogonal to mammalian cells, meaning they do not interact with any endogenous tRNAs or synthetases. We will evolve these pairs from existing tRNA-synthetase/suppressor-tRNA pairs using two approaches - """"""""cut - paste"""""""" and """"""""directed evolution"""""""". The newly generated mammalian cell can then synthesize large amounts of a homogeneous protein of interest containing an O-GlcNAc moiety at the desired sites. This unique and powerful technology can then be used to answer remaining questions about the mode of regulation of the many proteins that are known to undergo O-GlcNAcylation, such as the c-Myc protein. The c-Myc protein is an important oncogene that has been extensively studied. A complete understanding of its regulatory mechanism will be critical to the development of new therapies to treat or prevent cancer. It is known that c-Myc is modified with O-GlcNAc moiety only at one site (Thr-58). As a test of our system, we will investigate the effect of O-GlcNAcylation of Thr-58 of c-Myc on its protein stability, cellular localization and regulatory roles, especially the interplay between O-GlcNAcylation and phosphorylation at Thr-58. We will produce a series of homogeneous c-Myc proteins with different site-specific specific glycosylations and then performing various biological assays both in vivo and in vitro. These studies will shed light on the mechanism of regulation of proteins by glycosylation at a molecular level.
The long-term goal of this proposal is to study the regulatory role of O-N-acetylglucosamine (O-GlcNAc) modification in biological pathways. Comprehensive studies of the effects of O-GlcNAc as well as similar molecules on protein function have not been possible due to a number of technical obstacles. Yet, it is a highly significant area of research that is critical to our understanding of the functioning of living systems and biomedical research. As a starting point, I would like to probe the novel aspect of O-GlcNAc modification regulating oncogene c-Myc activities and elucidate its mechanism. One of the major bottlenecks for my project and related glycobiology is the inability to generate homogenous proteins bearing specific modifications in mammalian systems. I will circumvent these problems by hijacking the cell's translational machinery and generate new genetic codes or the non-natural glycosylated amino acids in mammalian cells. The newly developed mammalian cell will then synthesize a series of homogeneous glycosylated c-Myc proteins in vivo. This is a unique and powerful technology that can answer questions that could not be answered before such as nucleus relocation of c-Myc and interplay between phosphorylation and O-GlcNAcylation to regulate the ubiquitination and degradation of c-Myc. ? ? ?
