Mechanism-based traps enable protease and hydrolase substrate discovery

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.

Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

“…We identified two unreported Parkin substrates and validated their degradation by the ubiquitination-proteosome pathway. Very recently, a mechanism-based trap strategy was developed to discover hydrolase substrates by capturing the substrates with the enzyme 48 . While an extremely powerful approach, it is not suitable for profiling substrates involved in the formation of large complexes and requires the formation of a covalent bond between enzymes and substrates.…”

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation

“…Among the 20 proteins that showed enrichment in the RHBDL4-FLAG fraction greater than 1.4-fold were the chaperones BiP and calreticulin; two protein disulfide isomerases, namely PDI and Erp44; and both subunits of the regulatory glucosidase II (Table S2). Recently, it has also been shown that these soluble ER chaperones are cleaved by RHBDL4, promoting their secretion (Tang et al, 2022); however, the physiological relevance of this finding remains to be addressed. Furthermore, two homologous membrane-integral ERAD factors, namely Er-lin1 and Erlin2, were enriched by 1.5-fold.…”

Organism-wide secretome mapping uncovers pathways of tissue crosstalk in exercise

“…This C-terminus contains the ER retention signal required for intracellular localization of CES2. In addition, RHBDL4 has been proposed as a hydrolase that liberates an array of ER-resident proteins into the extracellular space (Tang et al, 2022). Consistent with this idea, we were unable to detect peptides corresponding to C-terminus of either CES2A or CES2C ( Fig.…”