A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation
The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase‐based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contai...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2019-07-01
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| Series: | FEBS Open Bio |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/2211-5463.12646 |
| _version_ | 1797681629365272576 |
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| author | Amin Tashakor Mahshid H‐Dehkordi Enda O'Connell Sergi Gomez Ganau Rafael Gozalbes Leif A. Eriksson Saman Hosseinkhani Howard O. Fearnhead |
| author_facet | Amin Tashakor Mahshid H‐Dehkordi Enda O'Connell Sergi Gomez Ganau Rafael Gozalbes Leif A. Eriksson Saman Hosseinkhani Howard O. Fearnhead |
| author_sort | Amin Tashakor |
| collection | DOAJ |
| description | The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase‐based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease‐Activating Factor‐1 (Apaf‐1) molecules and induces cell death by activating caspase‐9. Apaf‐1‐dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf‐1 fused to an N‐terminal fragment of luciferase binds to Apaf‐1 fused to a C‐terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill‐defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants. |
| first_indexed | 2024-03-11T23:47:38Z |
| format | Article |
| id | doaj.art-0cc73cfdeb554956b5fce424df076f3a |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-03-11T23:47:38Z |
| publishDate | 2019-07-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-0cc73cfdeb554956b5fce424df076f3a2023-09-19T08:50:32ZengWileyFEBS Open Bio2211-54632019-07-01971194120310.1002/2211-5463.12646A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formationAmin Tashakor0Mahshid H‐Dehkordi1Enda O'Connell2Sergi Gomez Ganau3Rafael Gozalbes4Leif A. Eriksson5Saman Hosseinkhani6Howard O. Fearnhead7Pharmacology and Therapeutics School of Medicine NUI Galway IrelandPharmacology and Therapeutics School of Medicine NUI Galway IrelandGenomics and Screening Core National Centre for Biomedical Engineering Science NUI Galway IrelandPROTOQSAR Valencia SpainPROTOQSAR Valencia SpainUniversity of Gothenburg SwedenTarbiat Modares University Tehran IranPharmacology and Therapeutics School of Medicine NUI Galway IrelandThe expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase‐based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease‐Activating Factor‐1 (Apaf‐1) molecules and induces cell death by activating caspase‐9. Apaf‐1‐dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf‐1 fused to an N‐terminal fragment of luciferase binds to Apaf‐1 fused to a C‐terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill‐defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.https://doi.org/10.1002/2211-5463.12646Apaf‐1apoptosomepentachlorophenolreproductive toxicityscreeningsplit luciferase complementation assay |
| spellingShingle | Amin Tashakor Mahshid H‐Dehkordi Enda O'Connell Sergi Gomez Ganau Rafael Gozalbes Leif A. Eriksson Saman Hosseinkhani Howard O. Fearnhead A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation FEBS Open Bio Apaf‐1 apoptosome pentachlorophenol reproductive toxicity screening split luciferase complementation assay |
| title | A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| title_full | A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| title_fullStr | A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| title_full_unstemmed | A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| title_short | A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| title_sort | new split luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation |
| topic | Apaf‐1 apoptosome pentachlorophenol reproductive toxicity screening split luciferase complementation assay |
| url | https://doi.org/10.1002/2211-5463.12646 |
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