Development of New Photocrosslinking Approaches to Discover Binding Partners of O-GlcNAc-Modified Proteins

dc.contributor.advisorConrad, Nicholasen
dc.contributor.committeeMemberKohler, Jennifer J.en
dc.contributor.committeeMemberFontoura, Beatrizen
dc.contributor.committeeMemberTu, Benjaminen
dc.creatorRodriguez, Andrea Christineen
dc.creator.orcid0000-0003-1457-8826 2015
dc.description.abstractO-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant post-translational modification that is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). While it is elusive how O-GlcNAc alters protein function, altered O-GlcNAc levels are associated with human diseases. To gain insight into the functional consequences of O-GlcNAc-ylation, we reported a method to incorporate the diazirine photocrosslinking group onto O-GlcNAc residues in cellular proteins. Photocrosslinking O-GlcNAc, O-GlcNDAz, yields covalent crosslinking between O-GlcNAc-ylated proteins and their binding partners, which further analysis can confirm these interactions. I applied the GlcNDAz technology to a heavily O-GlcNAc-modified nucleoporin NUP98 and NUP98 leukemogenic fusions, produced under chromosomal translocation, to gain insight into the mechanism of NUP98 fusion-mediated cell transformation in leukemia. The wild-type nucleoporins are associated with nuclear trafficking. In chapter 2, I demonstrated both NUP98 and NUP98 fusions are O-GlcNAc-modified. Additionally, evidence suggested O-GlcNAc is near the site of interaction based on crosslinking experiments. While a powerful approach, the utility of in-cell O-GlcNDAz crosslinking was restricted by several limitations. To solve this challenge, I first engineered a mutant OGA that is better able to remove GlcNDAz from proteins in order to facilitate homeostasis of O-GlcNDAz modification in cells and for potential use as an enzyme to release crosslinked material (chapter 3). Next, I constructed a mutant OGT to preferentially add GlcNDAz to proteins, in order to maximize possible crosslinking material (chapter 4). Finally, I initiated the development of a chemo-enzymatic synthesis of O-GlcNDAz-ylated peptides using three enzymes: A bifunctional enzyme consisting of Bifidobacterium longum N-acetylhexosamine 1-kinase (NahK) and E. coli N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) to generate UDP-GlcNDAz, then added GlcNDAz to peptides by human OGT (chapter 5). Together, these strategies allow for the development of a complementary cell free O-GlcNDAz crosslinking approach. O-GlcNDAz-modified peptides generated from this reaction can be crosslinked to molecules from cell lysates. This technology can be used to identify binding partners of O-GlcNAc-modified proteins, including both normal NUP98 and leukemogenic NUP98 fusions and may reveal functional roles of O-GlcNAc on NUPs and NUP fusions.en
dc.subjectMutation, Missenseen
dc.subjectNuclear Pore Complex Proteinsen
dc.titleDevelopment of New Photocrosslinking Approaches to Discover Binding Partners of O-GlcNAc-Modified Proteinsen
dc.type.materialtexten School of Biomedical Sciencesen Chemistryen Southwestern Medical Centeren of Philosophyen


Original bundle

Now showing 1 - 1 of 1
Thumbnail Image
22.74 MB
Adobe Portable Document Format

License bundle

Now showing 1 - 1 of 1
No Thumbnail Available
1.85 KB
Plain Text