Abstract
Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the TGN by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the ER. Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule sorting. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for sorting into secretory granules by self-aggregation.
Highlights
The contents of secretory granules are concentrated in a densely packed form leading to the dense-core appearance in electron microscopy
In Neuro-2a cells, aggregation of CC loop of prolactin (CCpN) and CCv was high, whereas it was reduced for CC loops from amylin (CCa) and even not statistically significant for CCr, CC loop of prolactin (CCpC), and again for CCc (Fig 2D)
In the case of provasopressin, granule sorting by self-aggregation of the wild-type protein and ER aggregation of diabetes insipidus mutant proteins were brought together by the amyloid hypothesis of secretory granule biogenesis proposed by Riek and colleagues (Maji et al, 2009; Beuret et al, 2017)
Summary
The constitutive secretory pathway continuously transports newly synthesized proteins to the plasma membrane and the extracellular space in all eukaryotic cells. Expression of prohormones and granins was shown to be sufficient to produce dense granule-like structures in fibroblasts and other cells lacking endocrine-specific machinery (Kim et al, 2001; Huh et al, 2003; Beuret et al, 2004) It is the specific environment of the TGN—reduced pH, high concentrations of Ca2+ and possibly other divalent cations, and the presence of glycosaminoglycans (Kolset et al, 2004; Dannies, 2012)—that triggers aggregation of regulated cargo proteins. Deletion of the glycopeptide and mutation of vasopressin inactivated ER aggregation of a folding-deficient NPII and strongly reduced sorting into granules and regulated secretion in AtT20 cells (Beuret et al, 2017) These results supported the aggregation model of granule biogenesis and suggested that the sequences that had evolved to mediate aggregation of the precursor at the TGN are responsible for pathological aggregation of mutant provasopressin in the ER to cause dominant disease. Our results support the hypothesis that CC loops in general contribute to granule sorting via self-aggregation
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