Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70

Abstract

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.

Figure 1

Ku70 is a granzyme A substrate. (A) Granzyme A (GzmA) cleaves Ku70, but not Ku80, in isolated nuclei. HeLa nuclei were incubated with indicated concentrations of GzmA or 1 μM S-AGzmA or GzmB at 37°C for indicated durations and analysed by immunoblot for Ku70, Ku80 or C23, a loading control. GzmA cuts Ku70 to generate a 35 kDa carboxy-terminal fragment, but Ku80 is not cleaved. Neither enzymatically inactive S-AGzmA nor GzmB cuts Ku70 or Ku80. (B) GzmA cuts recombinant Ku70. GzmA or inactive S-AGzmA was added to 0.5 μM Ku70 at 37°C for indicated durations and analysed by Ku70 immunoblot. The 35 kDa cleavage product is seen again. (C) GzmA and perforin (PFN) treatment of K562 degrades Ku70 and SET with similar kinetics. GzmA, but not GzmB or S-AGzmA, degrades Ku70 in cells. The 35 kDa cleavage fragment is also shown. (D) Ku70, like SET, is degraded in 40 min of cytotoxic T lymphocyte (CTL) attack. Ku70 and SET were not degraded when CTLs were pretreated with the GzmA inhibitor diisocoumarin. β-Actin is a loading control.

Figure 2

Granzyme A binds to Ku70 and Ku80 and disrupts Ku binding to DNA. (A) Ku70 and Ku80 in cell lysates bind to inactive S-AGzmA (GzmA, granzyme A). Precleared HeLa cell lysates were incubated with the indicated antibodies, and S-AGzmA and immune complexes were captured on protein G beads. GzmA binding was assayed using His-tag antibody. (B) Recombinant Ku70–GST (glutathione S-transferase) and Ku80–GST bind to S-AGzmA in vitro only in the presence of DNA. GST or Ku70–GST and Ku80–GST fusion proteins adsorbed on glutathione beads were incubated with S-AgzmA, with or without DNA. Even though the beads were treated with DNase I before extraction, S-AGzmA binding was detected with His-tag antibody only if DNA was present. (C) GzmA pretreatment blocks Ku complex association with DNA. Recombinant Ku70 and/or Ku80 that were either mock treated or preincubated with GzmA were incubated with a 600 bp DNA fragment and analysed for gel retardation by ethidium bromide staining. (D) Ku70 and Ku80 were pretreated with GzmA and then incubated with ³²P-labelled oligonucleotide and analysed by electrophoretic mobility shift assay. GzmA disrupts Ku binding to DNA at nanomolar concentrations.

Figure 3

Granzyme A (GzmA) disrupts the Ku complex in vivo. Ku complex staining declines in 15 min of adding GzmA and perforin (PFN) to HeLa cells, but Ku80 staining remains bright. DNA is stained with propidium iodide (PI). Control cells treated for 2 h with GzmA or PFN show no change in Ku complex staining. Apoptotic nuclear changes are readily apparent in 90 min after GzmA and PFN treatment.

Figure 4

Silencing Ku70 enhances granzyme A-induced DNA fragmentation and cell death, whereas overexpressing Ku70 decreases it. (A) HeLa cells treated with Ku70 short interfering RNA (siRNA), but not with green fluorescent protein (GFP) siRNA, have reduced Ku70 protein. A mixture of all four siRNAs, used in subsequent experiments, almost completely blocks Ku70 expression. Cells transfected with pcDNA6/V5His-Ku70 overexpress Ku70. Lysates were probed 3 days after transfection. (B) Ku70 protects against granzyme A (GzmA)-induced DNA damage. The mean±s.d. of three experiments is depicted. GzmA and perforin (PFN) cause more DNA damage in HeLa cells with silenced Ku70 and less damage in cells overexpressing Ku70. TUNEL, TdT-mediated dUTP nick end labelling. (C) Apoptosis was detected by annexin V and propidium iodide staining 1 h after adding GzmA and PFN. The mean±s.d. for three experiments is depicted.

Figure 5

Ku70 expression protects against cytotoxic T-lymphocyte-induced cell death. (A) Ku70 expression reduces target cell susceptibility to human lymphokine-activated killer attack. Transfected HeLa cells were attacked using an effector:target (E:T) ratio of 50:1. (B,C) Ku70^−/− mouse embryo fibroblasts (MEFs; open symbols) are more susceptible than Ku70^+/+ MEFs (filled symbols) to cytolysis (4 h ⁵¹Cr release) by gp33-specific cytotoxic T lymphocytes (CTLs) from T-cell receptor transgenic mice expressing all granzymes (P14 mice; B or P14 × GzmB^−/− mice; C). Targets were loaded with antigenic peptide (squares) or mock treated with medium (circles) before incubating with gp33-specific CTLs. P-values show significant differences at each E:T ratio.