Live-cell visualization of gasdermin D-driven pyroptotic cell death
| Autor: | George R. Dubyak, Joseph K. Rathkey, Derek W. Abbott, Jie Yang, Alex Yee-Chen Huang, Bryan L. Benson, Steven M. Chirieleison, Tsan Sam Xiao |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Programmed cell death Inflammasomes Recombinant Fusion Proteins Cell Immunology Mutagenesis (molecular biology technique) Biochemistry Models Biological 03 medical and health sciences Mice 0302 clinical medicine medicine Pyroptosis Animals Humans Point Mutation Molecular Biology Caspase Cell Line Transformed Innate immune system Microscopy Video biology Effector Macrophages Caspase 1 Intracellular Signaling Peptides and Proteins Cell Biology Phosphate-Binding Proteins Fusion protein Caspases Initiator Peptide Fragments Cell biology Neoplasm Proteins Luminescent Proteins Protein Transport 030104 developmental biology medicine.anatomical_structure HEK293 Cells Amino Acid Substitution Microscopy Fluorescence Caspases Proteolysis biology.protein Protein Multimerization 030217 neurology & neurosurgery |
| Popis: | Pyroptosis is a form of cell death important in defenses against pathogens, but which can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, gasdermin D (GSDMD), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and 11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis, but also enable its direct visualization. |
| Databáze: | OpenAIRE |
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