We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 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.Īctivity-based probes have confirmed the presence and selective reactivity of the catalytic serine or cysteine nucleophile for many hydrolase proteins in cells 6, 7, 8. In the first approach, substrates may be lost in the washing steps, and bound proteins may not be substrates.Ĭurrent approaches to identifying protease substrates mostly aim to either co-immunoprecipitate substrates that are non-covalently bound to catalytically inactive protease variants 10, 11, 12, or identify the peptides resulting from the action of the protease from experiments with and without the protease 13, 14, 15.Įfforts to define hydrolase specificity have captured non-covalent interactors with hydrolases and investigated the substrates that accumulate in the absence of a hydrolase or the products that accumulate in the presence of a hydrolase 9. The second approach typically underestimates the number of substrates, and the cleavages identified may be indirect 16, 17. The identification of intramembrane protease substrates by current approaches is particularly challenging 18, 19. The methodological challenges in defining hydrolase and protease specificity mean that the substrates of many proteases remain unknown or incomplete, and many hydrolases remain orphans-with unknown substrates and uncharacterized specificity. We previously demonstrated the genetically encoded, site-specific incorporation of photocaged Dap ((2 S)-2-amino-3-propanoic acid) (pc-Dap) into proteins expressed in Escherichia coli 2, 20. We converted pc-Dap to Dap (2,3-diaminopropionic acid) in proteins in vitro by illuminating purified proteins followed by incubation for up to 2 days at pH 8. By incubating purified proteases or thioesterases-in which we had replaced the catalytic cysteine or serine with Dap-with known substrates, we captured the otherwise transient thioester or ester intermediates-resulting from the first step of the reaction of these enzymes with their substrates-as their stable amide analogues. We demonstrated the utility of this approach for structural studies of acyl-enzyme intermediates 2.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |