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Exogenous Introduction of Initiator and Executioner Caspases Results in Different Apoptotic Outcomes.

ABSTRACT: The balance of pro-apoptotic and pro-survival proteins defines a cell's fate. These processes are controlled through an interdependent and finely tuned protein network that enables survival or leads to apoptotic cell death. The caspase family of proteases is central to this apoptotic network, with initiator and executioner caspases, and their interaction partners, regulating and executing apoptosis. In this work, we interrogate and modulate this network by exogenously introducing specific initiator or executioner caspase proteins. Each caspase is exogenously introduced using redox-responsive polymeric nanogels. Although caspase-3 might be expected to be the most effective due to the centrality of its role in apoptosis and its heightened catalytic efficiency relative to other family members, we observed that caspase-7 and caspase-9 are the most effective at inducing apoptotic cell death. By critically analyzing the introduced activity of the delivered caspase, the pattern of substrate cleavage, as well as the ability to activate endogenous caspases, we conclude that the efficacy of each caspase correlated with the levels of pro-survival factors that both directly and indirectly impact the introduced caspase. These findings lay the groundwork for developing methods for exogenous introduction of caspases as a therapeutic option that can be tuned to the apoptotic balance in a proliferating cell.

SUBMITTER: Anson F

PROVIDER: S-EPMC8385707 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundApoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides.MethodsThe full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays.ResultsThe transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). Transcriptional levels of RhCaspases 7, 8 and 9 in salivary glands increased more significantly than other tissues post-engorgement. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCaspase8 had no influence on tick feeding. Compared to the control group, apoptosis levels were significantly reduced after interfering with RhCaspase 7, 8 and 9 expressions. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase.ConclusionsTo the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.

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Exogenous Introduction of Initiator and Executioner Caspases Results in Different Apoptotic Outcomes.

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