Abstract
The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.
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Abbreviations
- hGrzB:
-
human granzyme B
- tBid:
-
truncated Bid
- KD:
-
knockdown
- MOMP:
-
mitochondrial outer membrane permeabilisation
- IAP:
-
inhibitor of apoptosis protein
- CTLs:
-
cytotoxic lymphocytes
- NK:
-
natural killer cell
- PCAF:
-
p300/CBP associated protein
- ADA3:
-
transcriptional adaptor 3
- PACS2:
-
phosphofurin acidic-cluster sorting-protein 2
- NS:
-
non-silencing
- AB:
-
Alamar blue
- NT:
-
non-targeting
- UV:
-
ultraviolet radiation
- CSP3:
-
caspase-3
- IP:
-
immunoprecipitation
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Acknowledgements
This study was supported by Program, Fellowship and Project grant support from the National Health and Medical Research Council (NHMRC) of Australia and the Cancer Council Victoria.
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Brasacchio, D., Noori, T., House, C. et al. A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage. Cell Death Differ 21, 748–760 (2014). https://doi.org/10.1038/cdd.2013.203
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DOI: https://doi.org/10.1038/cdd.2013.203
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