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dc.contributor.authorTang, Shan
dc.contributor.authorBeattie, Adam T
dc.contributor.authorKafkova, Lucie
dc.contributor.authorPetris, Gianluca
dc.contributor.authorHuguenin-Dezot, Nicolas
dc.contributor.authorFiedler, Marc
dc.contributor.authorFreeman, Matthew
dc.contributor.authorChin, Jason
dc.date.accessioned2022-03-21T02:02:25Z
dc.date.available2022-03-21T02:02:25Z
dc.date.issued2022-02
dc.identifier.issn0028-0836
dc.identifier.other35173328
dc.identifier.otherPMC8866121
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/335243
dc.description.abstractHydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.
dc.languageeng
dc.publisherSpringer Science and Business Media LLC
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourcenlmid: 0410462
dc.sourceessn: 1476-4687
dc.titleMechanism-based traps enable protease and hydrolase substrate discovery.
dc.typeArticle
dc.date.updated2022-03-21T02:02:22Z
prism.endingPage707
prism.issueIdentifier7898
prism.publicationNameNature
prism.startingPage701
prism.volume602
dc.identifier.doi10.17863/CAM.82674
dcterms.dateAccepted2022-01-07
rioxxterms.versionofrecord10.1038/s41586-022-04414-9
rioxxterms.versionVoR
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidTang, Shan [0000-0003-4483-1108]
dc.contributor.orcidPetris, Gianluca [0000-0002-2420-6359]
dc.contributor.orcidHuguenin-Dezot, Nicolas [0000-0001-8816-2159]
dc.contributor.orcidFiedler, Marc [0000-0003-4269-7873]
dc.contributor.orcidFreeman, Matthew [0000-0003-0410-5451]
dc.contributor.orcidChin, Jason [0000-0003-1219-4757]
dc.identifier.eissn1476-4687
pubs.funder-project-idMedical Research Council (MC_U105192713)
pubs.funder-project-idWellcome Trust (101035/Z/13/Z, 220887/Z/20/Z)
cam.issuedOnline2022-02-16


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International