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Mechanism-based traps enable protease and hydrolase substrate discovery.

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Researchers developed novel light-activated substrate traps to identify the functions of unknown hydrolase enzymes, including proteases. This method revealed new protease substrates and enzyme activities in human cells.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Hydrolase enzymes, including proteases, are crucial in biological processes, with a significant portion being drug targets.
  • The specific activities and substrate preferences of many hydrolases, particularly membrane-bound proteases, remain largely uncharacterized.
  • Understanding these enzymes is vital for drug discovery and deciphering cellular mechanisms.

Purpose of the Study:

  • To develop a novel strategy for identifying the substrates and activities of hydrolase enzymes.
  • To create mechanism-based, light-activated substrate traps for use in complex biological systems.
  • To uncover new substrates and functions for uncharacterized proteases and serine hydrolases.

Main Methods:

  • Engineered genetically encoded 2,3-diaminopropionic acid to replace catalytic nucleophiles (serine or cysteine) in hydrolases.
  • Developed light-activated substrate traps that form stable acyl-enzyme intermediates for substrate capture.
  • Applied these traps in complex mixtures and live mammalian cells to identify enzyme-substrate interactions.

Main Results:

  • Identified novel substrates for proteases, including the intramembrane protease RHBDL4 (Rhomboid Family Domain Containing 4).
  • Demonstrated RHBDL4's role in shedding luminal fragments of ER-resident type I transmembrane proteins and promoting chaperone secretion.
  • Characterized retinoblastoma binding protein 9 as an aminopeptidase with a preference for aromatic amino acid cleavage.

Conclusions:

  • The developed substrate trap strategy provides a powerful paradigm for discovering hydrolase substrates and activities.
  • This approach enables the functional characterization of previously unknown enzymes in complex biological contexts.
  • The findings expand our understanding of protease functions, particularly concerning membrane proteins and protein secretion pathways.