Abstract
Hydrolase 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.
Highlights
We previously demonstrated the genetically encoded, site-specific incorporation of photocaged Dap ((2S)-2-amino-3-{[(2-{[1(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl] thio}ethoxy)carbonyl]amino} propanoic acid) into proteins expressed in Escherichia coli[2,20]
Through stringent purification of substrates linked to high-temperature requirement protein A2 (HtrA2) protease in combination with a mass spectrometry workflow we identify more than 200 new substrates for this protease
We identify new substrates for the mammalian rhomboid protease RHBDL4, an intramembrane protease that resides in the endoplasmic reticulum
Summary
After illumination of cells and accumulated over time. We did not detect the conjugate by immunoblotting from cells expressing wild-type TEV, TEV(C151A) or cells expressing TEV(C151pc-Dap) before illumination. We detected Dap-specific conjugates 15 min after illumination of cells expressing RHBDL4(S144pc-Dap); these conjugates rapidly accumulated within 4 h (Extended Data Fig. 5d, Supplementary Fig. 10) Overall, these experiments demonstrated that we can express and optically activate a protease substrate trap for RHBDL4, and that the trap efficiently captures its model substrate. RHBDL4-mediated removal of the C-terminal ER retention motif from soluble ER-resident protein candidates was validated for protein disulfide-isomerases (protein disulfide-isomerase A6 (gene name: PDIA6) and ER protein 44 (gene name: ERP44) and calcium-binding chaperone Calreticulin (gene name: CALR), which were identified as potential substrates through our approach (Extended Data Fig. 8a–e). Additional tests confirmed that RHBDL4 cleaved other ER-resident soluble chaperones which were identified through our approach, including Calumenin, peptidyl-prolyl cis-trans isomerase FKBP9, Glucosidase 2 subunit α and β; in each case the cleavage led to secretion of the resulting N-terminal fragments into extracellular media (Supplementary Fig. 14). Unlike conventional secretases—which cleave transmembrane substrates in transmembrane domains or juxtamembrane domains to release ectodomains16,18—cleavage by RHBDL4 has the effect of removing the C-terminal ER-retention motif from a proportion of physiological a Pept(Dap-X)
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