Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models

TY - JOUR

T1 - Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models

AU - Beumer, Joep

AU - Bauzá-Martinez, Julia

AU - Veth, Tim S

AU - Geurts, Veerle

AU - Boot, Charelle

AU - Gilliam-Vigh, Hannah

AU - Poulsen, Steen S

AU - Knop, Filip K

AU - Wu, Wei

AU - Clevers, Hans

PY - 2022/11/16

Y1 - 2022/11/16

N2 - 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 proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.

AB - 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 proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.

KW - Humans

KW - Organoids

KW - Peptide Hydrolases/genetics

KW - Enteroendocrine Cells/metabolism

KW - Insulin/metabolism

KW - Endopeptidases/metabolism

U2 - 10.1073/pnas.2212057119

DO - 10.1073/pnas.2212057119

M3 - Article

C2 - 36343264

SN - 0027-8424

VL - 119

SP - e2212057119

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 46

ER -