5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 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 identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 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 bond 2 this enables stringent purification and identification of substrates. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile.
Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown.
Hydrolase enzymes, including proteases, are encoded by 2–3% of the genes in the human genome and 14% of these enzymes are active drug targets 1.
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