Discovery of AAA+ Protease Substrates through Trapping Approaches

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Proteases are crucial for maintaining cellular protein homeostasis (proteostasis).
• Identifying protease substrates and recognition mechanisms is challenging and requires experimental approaches.
• Understanding these interactions is key to cellular health and disease research.

Purpose of the Study:

• To review and summarize in vivo and in vitro substrate trapping methodologies for proteases.
• To highlight the biological outcomes and insights gained from published trapping studies.
• To provide recommendations for substrate trapping of specific chaperone-proteolytic systems.

Main Methods:

• In vivo genetic modifications to stabilize protease-substrate interactions.
• Affinity enrichment of trapped substrates followed by mass spectrometry (MS) identification.
• In vitro approaches using immobilized protease components and isolated cellular proteomes.

Main Results:

• Substrate trapping systematically identifies potential protease substrates, adaptors, and chaperones.
• Both in vivo and in vitro methods reveal substrate recognition signals (degrons) and conditional effects.
• Published studies demonstrate the utility of trapping for understanding proteostasis.

Conclusions:

• Substrate trapping is a powerful strategy for discovering protease substrates and their regulatory mechanisms.
• This review offers guidance for applying trapping techniques to AAA+ chaperone-proteolytic systems (Clp, Lon, FtsH).
• Further application of these methods will advance our understanding of cellular protein degradation pathways.