| The trefoil factor family (TFF) proteins play an essential role in the homeostasis of gastrointestinal (GI) surfaces, including healing after mucosal injury, and in GI cancers. TFF1 is an important tumor suppressor in the stomach, and TFF3 functions as a tumor promoter. They are two members of a unique family of peptides with a distinct three-loop structure, the trefoil domain, formed by a highly conserved motif of cysteine disulfide bonds.The overall goal of this project was to understand the structure-function relations of trefoil factors TFF1 and TFF3. I employed methods of bioinformatics, computational analyses of molecular structure and dynamics, recombinant protein production, direct structural measurements, as well as functional assays of cancer-associated cellular activities. The computational work focused on three specific areas:First, I studied the far Loop 1 edge of TFF1, which is the site of a cluster of human gastric cancer mutants, utilizing comparisons to related proteins. Models of the gastric cancer mutants were demonstrated to possess specific surface alterations without a disruption of the stability of the protein. Second, an analysis of sequence conservation and surface properties of TFF3 focused my attention on a pocket in the Loop 2/Loop 3 region. A number of mutations were chosen to test the functionality of this region, including residues in a sequence pattern resembling a well-recognized integrin-binding motif. Third, I evaluated the importance of the core cysteines to the stability of the trefoil domain. I performed molecular dynamics simulation of the cysteine mutants, which gave insight into the role of the cysteines.In parallel with the above computational work, I utilized molecular biology techniques and direct structural measurements, in order to pursue the above structure-based leads related to the mechanism of trefoil function. Some of these insights lead to important discoveries about the role of the TFF1 mutants in gastric cancer. Still others are being followed up at the time of this writing. This work laid the foundation of a larger project, setting the stage for the further investigation of the mechanisms and structural elements responsible for trefoil factor function. |
