Browsing by Subject "Mutation, Missense"
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Item Development of New Photocrosslinking Approaches to Discover Binding Partners of O-GlcNAc-Modified Proteins(2015-11-20) Rodriguez, Andrea Christine; Conrad, Nicholas; Kohler, Jennifer J.; Fontoura, Beatriz; Tu, BenjaminO-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.Item Missense Mutation in SCGN (Secretagogin) as a Possible Cause of Ulcerative Colitis in Three Siblings(2014-02-04) Llano, Ernesto; Burstein, Ezra; Chan, Lilliene; Harrison, Steven M.; Li, Haiying; Sifuentes-Dominguez, Luis; Park, Jason Y.; Cohen, Jonathan C.; Baker, Linda A.Inflammatory bowel disease (IBD) consists of two disorders, Crohn's disease and ulcerative colitis (UC), which affect 1.4 million Americans. Genetic factors contribute to the development of these disorders but not all culprit genes have been identified. To further our understanding of the genetic causes of IBD, we examined a consanguinous family with a high incidence of childhood-onset UC. We performed exome sequencing of five siblings, three of which were diagnosed with severe UC before the age of 10. Given the parents' consanguinity, areas of homozygosity were examined using a SNP array. Two areas of homozygosity, in chromosomes 6 and 12, were shared by the three affected probands but not by their two unaffected siblings. Given the inheritance pattern in the pedigree, we speculated that a homozygous recessive mutation in a gene contained within these intervals should be responsible for the phenotype. Thus, a total of 140 potential genes were implicated. Variants found by exome sequencing were prioritized if they affected the three probands and not their unaffected siblings, and if they were rare in the general population according to their frequency in the 1000 genomes database. Afterwards, we cross-referenced the identified potential culprit variants found in the exome sequence analysis against the unique areas of homozygosity shared by the probands. The top candidate change was in SCGN, which had a coding variant (c. 433G>A/p.R77H) not previously found in the 1000 genomes database or in the dbSNP database. Sanger sequencing confirmed that both parents were carriers, the three probands were homozygous, and one sibling was a carrier while the other was wild-type. SCGN encodes a 276 amino acid, calcium-binding protein, secretagogin. The protein is expressed in tissues of neuroendocrine lineage, such as pancreatic β-cells and intestinal enteroendocrine cells. The coding change found in these patients is located in one of the calcium binding domains (EF hand 2). Immunohistochemical analyses of the patients' colon biopsies did not demonstrate a decrease of the presence of secretagogin when compared to controls, suggesting that the mutation does not affect expression, but may have a functional effect yet to be determined. We are building a molecular and cellular model of the R77H mutation to identify if this coding change alters the function of the protein. If a functional impairment of SCGN is confirmed, it could represent the first description of enteroendocrine dysfunction playing a role in IBD pathophysiology.