Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y
Different programed cell death (PCD) modalities involve protein–protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either...
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MDPI AG
2023-02-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/13/2/297 |
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| author | Parisa Ghanavatian Hossein Salehi-Sedeh Farangis Ataei Saman Hosseinkhani |
| author_facet | Parisa Ghanavatian Hossein Salehi-Sedeh Farangis Ataei Saman Hosseinkhani |
| author_sort | Parisa Ghanavatian |
| collection | DOAJ |
| description | Different programed cell death (PCD) modalities involve protein–protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application. |
| first_indexed | 2024-03-11T09:05:28Z |
| format | Article |
| id | doaj.art-ec0b2c76470e4776bd82defc83ef9f1c |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-11T09:05:28Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-ec0b2c76470e4776bd82defc83ef9f1c2023-11-16T19:26:55ZengMDPI AGBiosensors2079-63742023-02-0113229710.3390/bios13020297Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5YParisa Ghanavatian0Hossein Salehi-Sedeh1Farangis Ataei2Saman Hosseinkhani3Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, IranDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, IranDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, IranDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, IranDifferent programed cell death (PCD) modalities involve protein–protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application.https://www.mdpi.com/2079-6374/13/2/297split luciferaseFADD/RIPK1 interactionRIPoptosomeSH-SY5Y cellsnecroptosis |
| spellingShingle | Parisa Ghanavatian Hossein Salehi-Sedeh Farangis Ataei Saman Hosseinkhani Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y Biosensors split luciferase FADD/RIPK1 interaction RIPoptosome SH-SY5Y cells necroptosis |
| title | Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y |
| title_full | Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y |
| title_fullStr | Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y |
| title_full_unstemmed | Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y |
| title_short | Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y |
| title_sort | bioluminescent ripoptosome assay for fadd ripk1 interaction based on split luciferase assay in a human neuroblastoma cell line sh sy5y |
| topic | split luciferase FADD/RIPK1 interaction RIPoptosome SH-SY5Y cells necroptosis |
| url | https://www.mdpi.com/2079-6374/13/2/297 |
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