Date of Award
Summer 8-22-2026
Document Type
Masters Thesis
Degree Name
M.S. in Biological Sciences
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
Cedric Asensio
Second Advisor
Xi Yang
Third Advisor
Joseph Angleson
Fourth Advisor
Jennifer Hoffman
Copyright Statement / License for Reuse
All Rights Reserved.
Keywords
Large dense-core vesicle (LDCV), HID-1, Protein binders, GFP-based assay, Regulated secretion
Abstract
The regulated secretory pathway is essential for hormone release and cellular homeostasis, yet the molecular mechanisms governing large dense-core vesicle (LDCV) biogenesis remain incompletely understood. HID-1 is a conserved trans-Golgi network protein required for regulated secretion and insulin granule maturation, but its mechanism of action remains unclear. This study investigated HID-1 function using two complementary approaches. First, computationally designed protein binders targeting HID-1 were generated using RFdiffusion, ProteinMPNN, and AlphaFold-Multimer. The binders were successfully expressed, purified, and fluorescently labeled, demonstrating the feasibility of developing non-antibody protein-labeling reagents, although HID-1 specificity requires further optimization. Second, a split GFP-based assay was developed to directly visualize HID-1 dimerization in living cells. Combined with secretion assays, localization studies, flow cytometry, and biochemical crosslinking, these experiments demonstrated that the disease-associated R432W mutation disrupts HID-1 dimerization and impairs regulated insulin secretion despite normal localization. Together, these findings identify HID-1 dimerization as a critical determinant of HID-1 function and establish novel tools for studying regulated secretion.
Copyright Date
8-2026
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Jenna E. Stainbrook
Provenance
Received from ProQuest
File Format
application/pdf
Language
English (eng)
Extent
82 pgs
File Size
1.2 MB
Recommended Citation
Stainbrook, Jenna E., "Characterization of HID-1 Self-Association and Its Role in Regulated Secretory Granule Function" (2026). Electronic Theses and Dissertations. 2805.
https://digitalcommons.du.edu/etd/2805