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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
Insights into the GSDMB-mediated cellular lysis and its targeting by IpaH7.8
Hang Yin1,2, Jian Zheng1,3, Qiuqiu He1,4
1The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Haihe Laboratory of Cell Ecosystem, Tianjin Institute of Immunology, Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, 300070, Tianjin, China.
Abstract:
The multifunctional GSDMB protein is an important molecule in human immunity. The pyroptotic and bactericidal activity of GSDMB is a host response to infection by the bacterial pathogen Shigella flexneri, which employs the virulence effector IpaH7.8 to ubiquitinate and target GSDMB for proteasome-dependent degradation. Furthermore, IpaH7.8 selectively targets human but not mouse GSDMD, suggesting a non-canonical mechanism of substrate selection. Here, we report the crystal structure of GSDMB in complex with IpaH7.8. Together with biochemical and functional studies, we identify the potential membrane engagement sites of GSDMB, revealing general and unique features of gasdermin proteins in membrane recognition. We further illuminate how IpaH7.8 interacts with GSDMB, and delineate the mechanism by which IpaH7.8 ubiquitinates and suppresses GSDMB. Notably, guided by our structural model, we demonstrate that two residues in the α1-α2 loop make the mouse GSDMD invulnerable to IpaH7.8-mediated degradation. These findings provide insights into the versatile functions of GSDMB, which could open new avenues for therapeutic interventions for diseases, including cancers and bacterial infections.
Insights
The gasdermin B (GSDMB) protein
Area of Science:
- Immunology
- Structural Biology
- Microbiology
Background:
- The gasdermin B (GSDMB) protein plays a crucial role in human immunity, exhibiting pyroptotic and bactericidal activities against bacterial pathogens like Shigella flexneri.
- Shigella flexneri utilizes the effector IpaH7.8 to ubiquitinate and degrade GSDMB, a process essential for bacterial virulence.
- IpaH7.8 exhibits selective substrate targeting, degrading human GSDMB but not mouse GSDMD, indicating a specific recognition mechanism.
Purpose of the Study:
- To elucidate the structural basis of the interaction between GSDMB and the Shigella effector IpaH7.8.
- To identify the mechanism by which IpaH7.8 ubiquitinates and suppresses GSDMB.
- To understand the structural determinants of IpaH7.8's substrate selectivity, particularly its differential targeting of human versus mouse gasdermin D (GSDMD).
Main Methods:
- X-ray crystallography to determine the structure of the GSDMB-IpaH7.8 complex.
- Biochemical assays to study protein interactions and ubiquitination.
- Functional assays to assess GSDMB activity and IpaH7.8-mediated degradation.
Main Results:
- The crystal structure of GSDMB in complex with IpaH7.8 was determined, revealing key interaction interfaces.
- Potential membrane engagement sites of GSDMB were identified, highlighting conserved and unique features among gasdermin proteins.
- The mechanism of IpaH7.8-mediated ubiquitination and suppression of GSDMB was elucidated, including the identification of specific residues involved in substrate recognition and degradation.
- Structural insights revealed that two residues in the α1-α2 loop of mouse GSDMD confer resistance to IpaH7.8-mediated degradation.
Conclusions:
- The study provides a detailed structural and mechanistic understanding of how Shigella flexneri targets GSDMB for degradation.
- Insights into gasdermin protein membrane recognition and IpaH7.8 substrate selectivity were gained.
- Findings offer potential therapeutic strategies for bacterial infections and cancers by targeting GSDMB-related pathways.
