PMC

PFP loading of GzmA or GzmB induces laminB cleavage. (a) GzmA or GzmB (1 μg) were loaded with PFP into K562 cells at 37°C for indicated times. Electrophoresed samples were probed with laminB mAb (101-B7), which recognizes full-length laminB and a 46-kDa cleaved product in cells treated with either GzmA or GzmB, as well as an unknown cross reactive protein migrating at 50 kDa. GzmA laminB cleavage was not blocked by preincubation with CI, maintained throughout the experiment. (b) GzmA loading of K562 cells generates a 25-kDa C-terminal laminB fragment at 90 min, visualized by using polyclonal Ab against the C terminus of laminB. Reprobing for the GzmA-interacting protein PHAPI () controlled for loading.

The nuclear lamina in K562 cells is disrupted after PFP-loading Gzms. K562 cells, mock-treated (A), or treated with PFP alone (B) or with PFP and GzmA (C–E) or GzmB (F and G) for 1 h, were stained with FITC-anti-laminB mAb 101-B7 (Left) or propidium iodide (Center); overlay (Right). Arrows in A and B indicate dissolution of the lamina and dispersion of laminB to the cytoplasm in mitotic cells. (C–E) Progressive changes in laminB degradation after GzmA treatment. Before chromatin changes are evident (C), lamin staining becomes punctate. As chromatin condensation and nuclear fragmentation progress, the nuclear lamina dissolves and lamin staining diminishes and disperses to the cytoplasm. K562 cells loaded with GzmB exhibit less pronounced changes in laminB staining. These changes persist even with CI (G).

LaminA/C are degraded in isolated HeLa nuclei by GzmA. GzmA-treated nuclei were analyzed by immunoblot probed with anti-laminA/C mAb and Ab to ref-1 as a control for loading. (a) LaminsA/C are degraded with similar kinetics as laminB (Fig. ). Four-fold dilutions of GzmA from 1.5 to 100 nM were added for the indicated times. Cleavage is blocked by GAI and is not induced by inactive S-AGzmA. GzmB does not induce laminA/C cleavage (not shown). (b) Ser protease inhibitors, but not CI, reduce GzmA cleavage of laminA/C. HeLa nuclei were incubated for 1 h with nothing (mock) or with 100 nM GzmA in the presence of no inhibitor, DMSO, or the indicated inhibitors. The percentage of cleavage in the presence of inhibitors compared to uninhibited enzyme is graphed. (c) LaminA/C cleavage is detected 90 min after PFP loading 4-fold dilutions of active GzmA (0.2–150 nM) into K562 cells.

LaminB-GST is cleaved in vitro by GzmA and GzmB. (a) LaminB (amino acids 143–551)-GST fusion protein was produced in Escherichia coli. Known sites of laminB cleavage by caspases after D231 and by the CRNSP after Y377 are indicated, as are the nuclear localization signal (NLS, underlined), and sites of phosphorylation by p34^cdc2 (S^† 383) and protein kinase C (S* 385, 395). Figure modified from ref. . (b) LaminB-GST protein (1 μg, black arrowhead) was incubated for 1 h at 37°C with 2-fold dilutions of GzmA (Left) or GzmB (Middle) beginning at a highest concentration of 1 μg and analyzed by SDS/PAGE and GelCode stain. Gzm bands are indicated by <. GzmA and GzmB generated cleavage fragments of laminB, indicated by white and gray arrowheads, respectively, were analyzed for N-terminal sequence. The * marks the 42-kDa N-terminal laminB fragment generated by GzmA; the N-terminal GzmB cleavage fragment was not visualized. The sequenced fragments contain the C terminus of the fusion protein because they stain with anti-GST by immunoblot (Right). The GzmA cleavage site is after R392 and the GzmB cleavage site is the known caspase site after D231. GzmA is more efficient than GzmB at cleaving laminB-GST.

LaminB is cleaved during CTL attack even in the presence of CI or bcl-2 overexpression. (a) Mouse peptide-specific CTL were incubated for the indicated times with an equal number of EL4 cells, preincubated with either medium or the cognate peptide. Lamin cleavage products expected for cleavage by GzmB (46 kDa, *) and GzmA (25 kDa, white arrowhead) are detected with Ab against the C terminus of laminB only in targets incubated with antigen. Although there is a background cross-reactive band in the target cells just below the expected 46-kDa caspase/GzmB fragment, the laminB cleavage signal is clearly enhanced in the presence of specific antigenic peptide. Additional minor laminB cleavage products of ≈42 kDa are also produced specifically during CTL attack. When target cells are preincubated with CI, maintained during the CTL attack, the cleavage products are still clearly seen. E and T indicate lysates of just effector CTL or target cells, respectively. (b) ConA-treated HeLa cells or HeLa cells overexpressing bcl-2 were incubated with human CMV-specific CTL and assayed for lamin cleavage by immunoblot and probed with polyclonal anti-laminB Ab or laminA/C. When granule exocytosis is blocked with EGTA, lamins are not cut. In the presence of Ca²⁺, both expected GzmA and caspase/GzmB fragments are detected. Bcl-2 overexpression does not inhibit lamin B cleavage and CI only partly reduces the appearance of the 46-kDa caspase/GzmB product. Bcl-2 and CI inhibit caspase-mediated laminA/C cleavage.

GzmA and GzmB cleave laminB in isolated nuclei independently of caspase activation. (a and b) Four-fold dilutions of GzmA ranging from 0.4 to 200 nM were added to isolated HeLa nuclei for the indicated times. Full-length laminB is indicated by a black arrowhead, the C-terminal 25-kDa laminB fragment by a white arrowhead, and the N-terminal 46-kDa fragment by a gray arrowhead. LaminB degradation requires the active Ser protease because S-AGzmA does not induce cleavage, and the reaction is blocked by GAI. (c) Ser protease inhibitors, but not caspase or other protease inhibitors, reduce lamin cleavage in HeLa nuclei by GzmA. HeLa nuclei were incubated for 1 h with nothing (mock) or with 100 nM GzmA in the presence of no inhibitor, DMSO, or the indicated inhibitors. Blot was reprobed for the nuclear protein Ref-1 as a loading control to quantitate enzymatic cleavage. The percentage of cleavage with inhibition was normalized to that with uninhibited enzyme. (d) GzmB induces laminB degradation in isolated nuclei less efficiently than GzmA. The 46-kDa C-terminal fragment after GzmB cleavage (*) is different from the 25-kDa fragment generated by GzmA cleavage (white arrowhead). Serial 4-fold dilutions of GzmA (starting with 200 nM) or GzmB (starting with 310 nM) were added for 2 h. LaminB is fragmented with 3 nM GzmA but requires at least 155 nM GzmB. (e) LaminB cleavage in GzmB-treated nuclei is not enhanced by adding cytosol (1 × 10 ⁷ cell equivalents at highest concentration followed by 4-fold serial dilutions) (Left). The Ser protease inhibitor DCI (lane 1), but not CI (lane 2) or DMSO (lane 3), blocks lamin cleavage by GzmB (Right). GzmB without inhibitors is shown in lane 4 and nuclei without GzmB in lane 5. Blot in a was probed with mAb 101-B7 and b--e with polyclonal laminB Ab.