Small disulfide loops in peptide hormones mediate self-aggregation in secretory granule biogenesis

Unlike constitutively secreted proteins, peptide hormones are first stored in densely packed secretory granules, before regulated release from the cell upon stimulation. Secretory granules are formed at the trans-Golgi network (TGN) by self-aggregation of the prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide (CC) loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of misfolding mutants in the endoplasmic reticulum (ER), proposing ER aggregates to be mis-localized amyloid aggregates that evolved to mediate sorting into granules. Several other hormone precursors also contain similar small CC loops suggesting their function as a general device to mediate aggregation for granule biogenesis.
To test this hypothesis, we studied the CC loops of various peptide hormones, vasopressin, amylin, calcitonin, prorenin, and prolactin, in order to determine their capacity (i) to induce ER aggregation of a misfolded reporter; (ii) to mediate aggregation of a constitutive protein into secretory granules.
For ER aggregation, CC loops were fused to a misfolded and truncated version of the neurophysin II portion of provasopressin, NPΔ, as a reporter, transiently expressed in COS-1 and Neuro-2a cells, and analyzed by immunofluorescence microscopy as well as by immunogold electron microscopy. We found that all CC loops induce ER aggregation although to different extents and with different morphologies, some only detectable by electron microscopy.
To study granule sorting, either one or two CC loops were fused to the constitutively secreted protein α-1 proteinase inhibitor (A1Pi), used as a reporter. Stably expressing AtT20 cell lines were generated and analyzed by immunofluorescence microscopy for accumulation in secretory granules, by stimulated secretion, and for insolubility to Lubrol extraction. All assays showed that the CC loops are able to reroute A1Pi into the regulated secretory pathway, thus supporting our hypothesis.
Altogether, the results show that CC loops mediate both the aggregation of a misfolded reporter in the ER as well as sorting of a constitutive protein into secretory granules, indicating that CC loops act as general secretory granule signals by mediating self-aggregation, advancing our mechanistic understanding of regulated secretion.