A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage

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.

Caught in the act – protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease

“…These findings suggest that myristoylation of tBid at Gly 61 may not be absolutely required for Bid action. Indeed, granzyme B, which cleaves human Bid at Asp ↓ -Ser 76 to generate a non-myristoylated Bid species, requires PACS-2 to recruit Bid to mitochondria (Li et al, 1998;Brasacchio et al, 2014). These findings suggest that following dephosphorylation, full-length Bid follows one of two distinct pathways leading to MOMP.…”

Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization

“…22 We recently showed that PCAF and ADA3 regulate the intrinsic cell death pathway through epigenetic regulation of PACS2, which in turn controls the subcellular trafficking of proapoptotic BID, the preferred target and substrate of hGrzB. 14,23 These findings led us to assess whether PCAF or ADA3 also control the expression of the related gene PACS1, whose encoded product is thought to regulate protein trafficking from the Golgi to the endosomal compartment. 21 Using targeted lentivirus shRNA, we found that depleting either PCAF or ADA3 in HeLa cells, or ADA3 in HCT-116 cells, significantly reduced PACS1 mRNA levels ( Figure 1a).…”

“…11,13 MOMP results in release of cytochrome c, SMAC/Diablo and Omi/HtrA2 to irreversibly activate a cascade of activated caspases. 14 In one critical pathway to mitochondrial-directed cell death, BID is processed either by caspase-3 at Asp60 or by the proapoptotic serine protease hGrzB at Asp75. 15,16 hGrzB is delivered from granules of cytotoxic lymphocytes through perforin (Pfn) pores that form in the target cell plasma membrane following conjugate formation between a killer cell and its target.…”

“…20 We recently reported that the mitochondrial pathway of apoptosis is regulated by p300/CBP-associated factor (PCAF) and human alteration/deficiency in activation 3 (ADA3), components of the epigenetic-modifying complexes ATAC and SAGA. 14 Moreover, reduced PCAF or ADA3 expression markedly reduces a cell's sensitivity to hGrzB by disrupting BID trafficking and reducing its availability for cleavage by hGrzB. 14 In turn, we traced cell protection to the decreased expression of phosphofurin acidic cluster sorting protein 2 (PACS2), a member of the PACS protein family known to regulate protein sorting and trafficking between subcellular secretory compartments.…”

“…14 Moreover, reduced PCAF or ADA3 expression markedly reduces a cell's sensitivity to hGrzB by disrupting BID trafficking and reducing its availability for cleavage by hGrzB. 14 In turn, we traced cell protection to the decreased expression of phosphofurin acidic cluster sorting protein 2 (PACS2), a member of the PACS protein family known to regulate protein sorting and trafficking between subcellular secretory compartments. 21 We now report that PCAF and ADA3 also transcriptionally regulate the related gene PACS1, whose protein product is reported to control protein traffic between the trans-Golgi network (TGN) and the endosomal compartment.…”