A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage.
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.
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Brasacchio
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Noori
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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House
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Brennan
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Simpson
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia [3] The Beatson Institute for Cancer Research, Glasgow, UK.
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Susanto
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia.
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Bird
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Therapeutics Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Johnstone
1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia [2] Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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Trapani
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D023281:Genomics / Q000379:methods; D053804:Granzymes / Q000378:metabolism* / Q000494:pharmacology; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D054353:Perforin / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D050880:p300-CBP Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000099224:p300-CBP-Associated Factor
D051023:BH3 Interacting Domain Death Agonist Protein; D053804:Granzymes; D054353:Perforin; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C494814:BID protein, human; C444103:TADA3 protein, human