Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis
| Autor: | She Chen, Yuyong Ma, Andrea N. Johnston, Shuzhen Liu, Hua Liu, Zhigao Wang, Chuo Chen, Sarah Hanna-Addams |
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| Rok vydání: | 2020 |
| Předmět: |
Necroptosis
Mutant Cell Line Proinflammatory cytokine Small Molecule Libraries Cell membrane Piperidines Protein Domains Heat shock protein medicine Animals Humans HSP70 Heat-Shock Proteins Binding Sites Multidisciplinary Molecular Structure Chemistry Biological Sciences Hsp70 Cell biology Cell killing medicine.anatomical_structure Gene Knockdown Techniques Mutation Protein Multimerization HT29 Cells Protein Kinases Protein Binding Cysteine |
| Zdroj: | Proc Natl Acad Sci U S A |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1916503117 |
| Popis: | Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro, recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis. |
| Databáze: | OpenAIRE |
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