From Condensation to Complexity: The Multifaceted Roles of Chromogranin B in Secretory Protein Sorting via Molecular Condensation
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cell Biology
First Advisor
von Blume, Julia
Abstract
The biogenesis of secretory storage granules within the trans-Golgi network (TGN)is a fundamental process for the regulated secretion of proteins essential for maintaining cellular and physiological equilibrium. Among these proteins, the packaging and secretion of insulin is paramount for metabolic homeostasis. Despite the recognized role of Chromogranin B (CGB) in facilitating the process, the detailed mechanisms underlying its action within the secretory pathway have remained elusive. In my thesis, we explored the role of CGB in the biogenesis of secretory storage granules, with a focus on insulin secretion in pancreatic ? cells, a critical aspect of diabetes pathology. Utilizing a combination of biochemical assays and live-cell imaging, we uncover that CGB undergoes pH-dependent condensation within the TGN, serving as a pivotal scaffold for protein recruitment. This process is independent of traditional sorting receptors, suggesting a novel, receptor-independent pathway for granule biogenesis. Knockout studies further demonstrated the indispensable role of CGB in regulating insulin granule formation and secretion, signifying the essential role of CGB in the regulated secretory pathway. Employing a combination of biochemical and biophysical analysis and molecular dynamics simulations, we also elucidate the molecular mechanisms underlying CGBinduced condensation and its implications for granule formation and insulin biosynthesis. We reveal that intrinsically disordered regions (IDRs), electrostatic interactions, and a distinctive disulfide-bonded loop are key to its condensation behavior and granule formation. Electrostatic interactions and histidine residues confer CGB multivalency and pH sensitivity in molecular condensation. Molecular dynamic simulations and in vitro analysis provide additional insights into the disulfide-bonded loop of CGB, underscoring its role in the functional assembly of CGB. These findings demonstrate how the biophysical properties enable CGB to undergo phase separation under physiological conditions, thus facilitating its function as a scaffold for protein recruitment at the TGN. We also explore the selectivity of CGB condensate-dependent protein sorting. Besides the abundance of cargo proteins in the TGN, the protein size and the presence of the disulfide-bonded loops significantly influence the sorting and packaging into the secretory granules. This selectivity underscores a complex mechanism of protein recruitment, suggesting the importance of specific protein interactions between CGB condensate and clients for efficient sorting into secretory granules. Together, these studies propose a model that elucidates a novel non-receptormediated mechanism of protein sorting based on the multifaceted nature of CGB condensation in regulated secretion. This model encompasses the role of CGB condensation in granule biogenesis, protein recruitment, sorting, packaging, and potential role in interacting with the TGN membrane and releasing cargo. It offers a comprehensive view of CGB's critical role in ensuring the precise and efficient processing of secretory proteins, such as insulin, within the cellular secretory pathway. Our findings contribute to a deeper understanding of the cellular mechanisms governing hormone storage and release, laying the groundwork for future research aimed at unraveling the complexities of regulated protein secretion and its implications for diseases associated with secretion dysfunction.
Recommended Citation
Tian, Meng, "From Condensation to Complexity: The Multifaceted Roles of Chromogranin B in Secretory Protein Sorting via Molecular Condensation" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1416.
https://elischolar.library.yale.edu/gsas_dissertations/1416
