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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
Identification of SERPINB1 as a physiological inhibitor of human granzyme H
Li Wang1, Qian Li, Lianfeng Wu
1Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Abstract:
The granzyme/perforin pathway is a major mechanism for cytotoxic lymphocytes to eliminate virus-infected and tumor cells. The balance between activation and inhibition of the proteolytic cascade must be tightly controlled to avoid self damage. Granzyme H (GzmH) is constitutively expressed in NK cells and induces target cell death; however, how GzmH activity is regulated remains elusive. We reported earlier the crystal structures of inactive D102N-GzmH alone and in complex with its synthetic substrate and inhibitor, as well as defined the mechanisms of substrate recognition and enzymatic activation. In this study, we identified SERPINB1 as a potent intracellular inhibitor for GzmH. Upon cleavage of the reactive center loop at Phe(343), SERPINB1 forms an SDS-stable covalent complex with GzmH. SERPINB1 overexpression suppresses GzmH- or LAK cell-mediated cytotoxicity. We determined the crystal structures of active GzmH and SERPINB1 (LM-DD mutant) in the native conformation to 3.0- and 2.9-Å resolution, respectively. Molecular modeling reveals the possible conformational changes in GzmH for the suicide inhibition. Our findings provide new insights into the inhibitory mechanism of SERPINB1 against human GzmH.
Insights
This study identifies SERPINB1 as a key inhibitor of Granzyme H (GzmH), a protein crucial for cytotoxic lymphocytes. SERPINB1 regulates GzmH activity, preventing potential self-damage and controlling cell death pathways.
Area of Science:
- Immunology
- Molecular Biology
- Structural Biology
Background:
- The granzyme/perforin pathway is essential for cytotoxic lymphocytes to eliminate infected or cancerous cells.
- Regulation of this pathway is critical to prevent self-inflicted damage.
- Granzyme H (GzmH) is involved in target cell death, but its regulation is not fully understood.
Purpose of the Study:
- To identify and characterize intracellular inhibitors of Granzyme H (GzmH).
- To elucidate the mechanism by which SERPINB1 inhibits GzmH activity.
Main Methods:
- Protein crystallography to determine the structures of GzmH and SERPINB1.
- Biochemical assays to analyze the interaction between GzmH and SERPINB1.
- Molecular modeling to understand conformational changes during inhibition.
Main Results:
- SERPINB1 was identified as a potent intracellular inhibitor of GzmH.
- SERPINB1 forms a stable covalent complex with GzmH upon cleavage of its reactive center loop.
- SERPINB1 overexpression reduced GzmH-mediated cytotoxicity.
- Crystal structures revealed the native conformations of GzmH and SERPINB1, providing insights into the inhibition mechanism.
Conclusions:
- SERPINB1 acts as a suicide inhibitor of GzmH.
- This finding provides a deeper understanding of GzmH regulation within the granzyme/perforin pathway.
- The study offers insights into controlling cytotoxic lymphocyte activity.
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