Optimised proteomic analysis of insulin granules from MIN6 cells identifies Scamp3, a novel regulator of insulin secretion and content.

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posted on 2024-09-25, 15:51 authored by Nicholas Norris, Belinda Yau, Carlo Famularo, Hayley Webster, Thomas Loudovaris, Helen E. Thomas, Mark Larance, Alistair M. Senior, Melkam A. Kebede

Abstract

Pancreatic b-cells in the Islets of Langerhans are key to maintaining glucose homeostasis, by secreting the peptide hormone insulin. Insulin is packaged within vesicles named insulin secretory granules (ISGs), that have recently been considered to have intrinsic structures and proteins that regulate insulin granule maturation, trafficking, and secretion. Previously, studies have identified a handful of novel ISG-associated proteins using different separation techniques. Here, this study combines an optimized ISG isolation technique and mass spectrometry-based proteomics, with an unbiased protein correlation profiling and targeted machine learning approach to uncover 211 ISG-associated proteins with confidence. Four of these proteins: Syntaxin-7, Synaptophysin, Synaptotagmin-13 and Scamp3 have not been previously ISG-associated. Through colocalization analysis of confocal imaging we validate the association of these proteins to the ISG in MIN6 and human b-cells. We further validate the role for one (Scamp3) in regulating insulin content and secretion from b-cells for the first time. Scamp3 knock-down INS-1 cells show a reduction in insulin content and dysfunctional insulin secretion. These data provide the basis for future investigation of Scamp3 in b-cell biology and the regulation of insulin secretion.

Keywords: insulin secretory granule, beta-cell, pancreas, proteomics, syntaxin-7, synaptophysin, synaptotagmin-13, scamp3

Article Highlights

· This study optimizes insulin granule isolation techniques alongside enhanced proteomics analyses to establish the first murine insulin secretory granule proteome.

· We investigated what proteins are present on insulin granules from MIN6 cells to further our understanding of insulin granule biogenesis, trafficking, and secretion.

· We found 211 insulin granule-associated proteins and validated 4 novel proteins.

· Through further functional studies, we implicated Scamp3 as a novel protein that regulates insulin content and secretion in b-cells.