Mutant Secretagogin, a Potential Cause of Ulcerative Colitis, Exhibits Reduced Affinity for SNARE Complex Protein SNAP-25

Date

2016-01-19

Authors

Norris, Nicholas

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Abstract

INTRODUCTION: Inflammatory Bowel Diseases (IBD) encompass a set of poorly understood multifactorial inflammatory disorders including Chrohn's disease (CD) and Ulcerative Colitis (UC). To date, around 200 genetic variants associated with increased risk of IBD have been identified through genome-wide association studies. Recently, our lab identified three siblings with UC from consanguineous parents who were all homozygous for a rare hypomorphic variant in the gene SCGN which encodes the calcium sensing protein secretagogin. Within the GI tract, secretagogin is localized specifically to enteroendocrine cells (EECs) suggesting an unforeseen role of EECs in IBD pathogenesis. This disease associated variant results in an amino acid substitution (R77H) in the Ca2+ binding region of secretagogin, which has been shown to interact with SNARE proteins in a Ca2+ dependent manner to mediate membrane fusion and exocytosis. Therefore, we examined whether this SCGN mutation impairs its Ca2+ dependent binding to the SNARE complex component SNAP-25 in vitro. METHODS: A GST pull-down assay was performed using recombinant GST fusion proteins of human secretagogin (WT or R77H) immobilized to glutathione-agarose beads. These beads were used to affinity purify SNAP-25 from whole cell lysates of STC-1 cells, a mouse enteroendocrine tumor cell line. Because SNAP-25 interactions with secretagogin are Ca2+ dependent, whole cell lysates were prepared in either Ca2+ rich or Ca2+ free conditions. The beads were washed and bound proteins were detected by SDS-PAGE and western blot analysis using appropriate antibodies. RESULTS: Under Ca2+ conditions, SNAP-25 was successfully bound to secretagogin, but the amount of SNAP-25 bound to mutant R77H secretagogin was significantly less. As expected, SNAP-25 did not bind secretagogin under Ca2+ free conditions. CONCLUSIONS: We confirm secretagogin interacts with SNAP-25 in a Ca2+ dependent fashion and the R77H mutation, located in the Ca2+ binding pocket of secretagogin, disrupts this interaction representing a hypomorphic mutation. This finding is consistent with the recessive nature of this mutation and strengthens its physiological relevance. Furthermore, it suggests that impaired exocytosis and hormone release from EECs contributes to the disease pathogenesis. Further study to elucidate which (if any) EEC secretory products play key roles in IBD prevention is needed. Understanding potentially aberrant paracrine mechanisms underlying IBD could open the door to novel treatments like hormone replacement therapy.

General Notes

The 54th Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 19, 2016, 2-5 p.m., D1.700)

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Citation

Norris, N., Sifuentes-Dominguez, L., & Burstein, E. (2016, January 19). Mutant secretagogin, a potential cause of ulcerative colitis, exhibits reduced affinity for SNARE complex protein SNAP-25. Poster presented at the 54th Annual Medical Student Research Forum, Dallas, TX. Retrieved from https://hdl.handle.net/2152.5/3257

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