The human brm protein is cleaved during apoptosis: the role of cathepsin G

Abstract

The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation. We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis. In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates. In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis. In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP). Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm. This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.

Figure 1

The brm and PARP proteins are degraded in NB4 cells undergoing apoptosis. (A) NB4 cells were treated with 100 J/m² of UV and then lysed at the times after treatment indicated above the lanes. Cell lysates were used for Western blotting with anti-brm and anti-PARP antibodies. (B) Cells were treated for 2 h with etoposide, camptothecin, or staurosporine at the indicated concentrations and then lysed for Western blotting.

Figure 2

Incubation of HeLa cell nuclei with purified, active caspases and cathepsins. (A) HeLa cell nuclei were prepared and resuspended in 100 μl of homogenization buffer as described in Materials and Methods. Then 100 ng of purified, active caspase-3 or caspase-7 was added to each 100-μl sample, and the samples were incubated at 30°C for the times indicated above the lanes. Control samples were incubated 2 h at 30°C without added caspase. Immediately after incubation, nuclei were repelleted and then lysed in a small volume of Nonidet P-40 lysis buffer for 30 min at 4°C. The Nonidet P-40 lysates were cleared of DNA by spinning 10 min at 10,000 × g and then used for Western blotting. (B) HeLa nuclei were incubated at 30°C for 2 h in buffer, HeLa cytosol, or NB4 cytosol, as indicated above the lanes. (C) Nuclei were incubated with buffer, purified cathepsin G, cathepsin D, or elastin as indicated.

Figure 3

Effect of serine protease inhibitors on cleavage of hbrm and activation of caspases. NB4 cells were left untreated, pretreated for 30 min with 5 μM cathepsin G inhibitor CK-08 (Z-Gly-Leu-Phe-CK), or pretreated for 30 min with the more general serine protease inhibitor l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) as indicated above the lanes. Untreated and inhibitor-treated samples were then UV-irradiated and incubated a further 4 h (with protease inhibitors added back to the appropriate samples). Whole cell lysates were then prepared as described in Materials and Methods and used for Western blot analysis with antibodies to the proteins indicated at left.

Figure 4

Endogenous murine brm cleavage is enhanced by over-expression of human cathepsin G in murine 32D cells. (A) Murine 32D cells stably transfected with either empty vector or vector expressing human cathepsin G were UV-irradiated and then lysed at the times after irradiation indicated above the lanes. Cell lysates were then used for Western blot analysis with anti-cathepsin G antibodies. (B) 32D lysates from the same experiment shown in A were used for Western blot analysis with anti-hbrm antibodies. Positions of full-length endogenous murine brm (180 kDa) and cleavage product (160 kDa) are indicated.

Figure 5

Staining of NB4 and HeLa-S3 cells with anti- cathepsin G antibodies or LysoTracker. (A) NB4 cells were incubated for 1 h at 37°C in media containing 50 μM LysoTracker peptide and then spun onto slides by using a cytocentrifuge. After fixation in neutral-buffered formalin followed by washing, the cells were incubated first with anti-cathepsin G antibodies and then with FITC-labeled secondary antibodies. Cells were prepared for viewing under the microscope by adding mounting media containing DAPI and coverslips. Photographs show DAPI plus cathepsin G staining, DAPI plus LysoTracker, DAPI alone, cathepsin G alone, or LysoTracker alone, as indicated. (B) HeLa-S3 cells were prepared as in A but were not incubated with LysoTracker. (C) NB4 cells were UV-irradiated as described in Materials and Methods; 1 h after irradiation, 50 μM LysoTracker was added to the cell media. After an additional hour of incubation, cells were spun onto a glass slide. After fixing and staining with anti-cathepsin G antibodies, cells were mounted by using DAPI-containing medium. Cell shape and chromatin condensation was used to distinguish apoptotic from nonapoptotic cells.

Figure 6

The proteasome is involved in hbrm degradation. (A) NB4 cells or COS-7 cells were treated with 20 μM lactacystin for 4 h or left untreated. Cells were then lysed for Western analysis. (B) COS-7 cells were transfected with either HA-tagged hbrm plasmid plus empty vector or with HA-tagged hbrm plasmid plus vector expressing 6-His Ubiquitin, as indicated above the lanes. Both cell samples were lysed, incubated with Ni-nitrilotriacetic acid agarose for binding of 6-His-tagged proteins, and then bound proteins eluted with 150 mM EDTA. Eluted proteins were used for Western blot analysis with anti-HA tag antibodies.

Figure 7

The 160-kDa hbrm cleavage fragment is less strongly associated with other nuclear proteins than full-length hbrm. (A) Diagram of hbrm domains and cathepsin G cleavage site. (B) NB4 cells were irradiated with UV, and then at various times after irradation the cells were fractionated into cytosol and nuclei by rapid detergent lysis (see Materials and Methods). Both fractions were used for Western blot analysis with anti-hbrm and anti-PARP antibodies.