Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis

Area of Science:

• Cellular biology
• Molecular mechanisms of cell death
• Protein-protein interactions

Background:

• Necroptosis is a regulated form of necrotic cell death implicated in various diseases.
• The signaling pathway involves the formation of mixed lineage kinase domain-like protein (MLKL) polymers, leading to cell membrane disruption.
• Understanding the molecular players in necroptosis is crucial for therapeutic development.

Purpose of the Study:

• To identify small molecules that inhibit necroptosis.
• To elucidate the role of heat shock protein 70 (Hsp70) in the necroptosis pathway.
• To characterize the interaction between Hsp70 and MLKL.

Main Methods:

• Screening of compound libraries to identify necroptosis inhibitors.
• Biochemical assays to study protein interactions and polymerization.
• In vitro studies using recombinant proteins and cell-based assays with knockdown and mutants.
• Chemical conjugation analysis of NBC1 with Hsp70.

Main Results:

• Necroptosis-blocking compound 1 (NBC1) was identified as a small-molecule inhibitor of necroptosis that conjugates to Hsp70.
• Hsp70 interacts with MLKL's N-terminal domain and promotes its polymerization via its substrate-binding domain (SBD).
• NBC1 inhibits Hsp70's SBD function by covalently modifying cysteines 574 and 603, blocking MLKL polymerization.
• Hsp70 knockdown destabilizes MLKL, suggesting it acts as a molecular chaperone for MLKL.

Conclusions:

• Hsp70 plays a dual role in necroptosis: stabilizing MLKL under normal conditions and promoting MLKL polymerization during necroptosis.
• The substrate-binding domain of Hsp70 is critical for promoting MLKL polymerization.
• NBC1 is a valuable tool for studying necroptosis and targeting Hsp70 function.