An integral atlas of hormone peptides of human EECs and their dedicated proteases

Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large pre-proteins that undergo proteolytic processing through peptidases to generate bioactive peptides. Due to the rarity of EECs and lack of cell models, prohormone processing remains challenging to assess. Here, we generated a panel of human organoid models in which EECs can efficiently be induced, combined with CRISPR-Cas9-mediated genetic inactivation or alteration of peptidases. We employed mass spectrometry based analysis to monitor peptide processing in knock-outs and wild type organoids for various hormone-processing enzymes (including PCSK1, PCSK2, PCSK1N, PCSK1mut_S375G, PCSK2mut_R430W, DPP4, CPB1 and CPE). We identify PCSK2-dependent Glucagon production in EECs, which is stimulated by activation of BMP signaling. We further map the substrates of other major EECs endo- and exopeptidases.