A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation

• Published in: FEBS open bio Vol. 9; no. 7; pp. 1194 - 1203
• Main Authors: Tashakor, Amin, H‐Dehkordi, Mahshid, O'Connell, Enda, Gomez Ganau, Sergi, Gozalbes, Rafael, Eriksson, Leif A., Hosseinkhani, Saman, Fearnhead, Howard O.
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01-07-2019; John Wiley and Sons Inc; Wiley
• Subjects: Adenosine triphosphate; Apaf-1; Apoptosis; Apoptosis - drug effects; apoptosome; Apoptosomes - drug effects; Apoptosomes - metabolism; Apoptotic Protease-Activating Factor 1 - metabolism; Biochemistry & Molecular Biology; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Caspase; caspase activation; ced-4; Cell Death; Cytochrome; Cytochrome c; Cytochromes c - metabolism; extracts; Fertility; Glycerol; HEK293 Cells; Humans; Luciferases - metabolism; pathways; Pentachlorophenol; Pentachlorophenol - pharmacology; Pentachlorophenol - toxicity; Preservatives; Proteins; reproductive toxicity; requirement; screening; Signal Transduction; Small Molecule Libraries; Spermatogenesis; split luciferase complementation assay; Toxicants; Toxicity; Toxicity Tests - methods; Wood preservatives; United States > US; screening; Apaf-1; apoptosome; split luciferase complementation assay; pentachlorophenol; reproductive toxicity
• DOI: 10.1002/2211-5463.12646
• EISSN: 2211-5463

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.
Oligomerization of Apoptotic Protease‐Activating Factor‐1 fused to luciferase fragments reconstitutes luciferase activity and generates light during apoptosome formation. Here, this luciferase‐based assay was used to screen a toxicant library. Pentachlorophenol was identified as an inhibitor of apoptosome formation, and subsequent experiments showed it acts by directly targeting cytochrome c.