Munc13-4 Regulates Peptide Hormone Degradation in Neuroendocrine Cells

项目总结

PROJECT SUMMARY Peptide hormone biosynthesis in the RER, packaging into secretory granules (SGs) in the Golgi, and exocytosis of SGs at the plasma membrane are well-studied processes in neuroendocrine cells. However, there is limited understanding of intracellular peptide hormone degradation and SG turnover. Only a small fraction (<10%) of SG-stored peptide hormones are utilized in physiological stimulation so that most SGs would undergo turnover. SG degradation mechanisms need to be balanced by SG biogenesis to maintain SG pools it is unclear how this coordination is achieved. Classical EM studies in the pituitary and endocrine pancreas had shown that SGs merge with multivesicular body (MVB)/lysosomes for degradation in a process termed crinophagy. We developed several fluorescence-based cell assays in neuroendocrine BON cells to detect the SG-MVB/lysosome merge. Because Munc13-4 is involved in endosomal fusion mechanisms in multiple cell types, we conducted the assays in cells knocked down for Munc13-4 and observed strong inhibition. In aim 1, we will extend structured illumination microsope studies to resolve whether crinophagy is the major mechanism and whether it is accompanied by microautophagy. We will pursue CgA timer protein studies to document that older SGs are preferentially degraded and determine whether such SGs are marked by ubiquitination. Lastly, by reconstituting SG-MVB/lysosome merge in a cell-free assay, we will determine whether Munc13-4 acts directly to promote the SG-MVB/lysosome merge. In efforts to document that Munc13-4 knockdown should mimic lysosome inhibitor treatment, we revealed that Munc13-4 knockdown has additional effects likely upstream from SG degradation. Instead of SG accumulation, we observed that SGs were smaller. When examined carefully, the small SGs were observed to undergo constitutive exocytosis. This implies that Munc13-4 also has effects on the SG biogenesis pathway. Follow-up studies will include immunofluorescense studies of marker proteins to determine where small SGs emerged in Munc13-4 knockdown cells. The overall phenotype of Munc13-4 knockdown is dominated by a shift from SG degradation to constitutive exocytosis. Were this to be true for hormones or prohormones this would have physiological consequences that we will assess in a Munc13-4 KO mouse. In aim 2, we will determine the mechanistic basis for the Munc13-4 regulation of the SG-MVB/lysosome merge. An siRNA screen using a robust fluorescence-based assay for the SG-MVB/lysosome merge, we will identify Rab, SNARE, and accessory factors required for SG-MVB fusion. We will develop a liposome-based fusion assay using the proteins identified in the screen. This will allow us to test what factors like Rab27 regulate Munc13-4-dependent fusion. Lastly, with highly purified Munc13-4 protein we will determine its structure by cryo-EM. Based on a on a liposome fusion assay, we will determine the tethering properties of Munc13-4 in cryo-ET studies. Overall this work will reveal important new cellular roles for Munc13-4 in neuroendocrine cells and contribute a molecular understanding on the tethering role for Munc13-4.