IntroductionGranzyme H (GzmH) is regarded as an orphan granzyme with unknown biologic functions in immune defense cells. 1 Recent reports show that this serine protease is predominantly expressed at high levels in natural killer (NK) cells and has chymotrypsin-like (chymase) activity. 2 No functional studies have as yet been reported. Granzymes (granule enzymes) are of particular interest due to their different proteolytic specificities and potential abilities to trigger cell death in tumor and virally infected cells. Indeed GzmA, a "tryptase" that cleaves after basic residues, and GzmB, an "aspase" preferring acidic residues, have evolved distinct apoptotic pathways. The cytotoxic action of GzmB on target cells is largely caspase dependent, 3 while that of GzmA is caspase independent. 4 In particular, GzmA, transferred into target cells via perforin, leads to a very fast generation of reactive oxygen species (ROS) and induces the translocation of the previously described endoplasmic reticulum (ER)-associated suppressor of variegation, enhancer of zeste, and trithorax (SET) complex into the nucleus. There, GzmA relieves the active nuclease NM23-H1 from the complex by destroying its bound inhibitor SET. 5 Recently, a caspaseindependent cell-death mechanism was also observed for murine GzmC, which rapidly induced mitochondrial swelling and membrane depolarization. 6 GzmM, which cleaves after residues with long, uncharged side chains such as methionine and leucine, 7 was also shown to induce a very rapid form of caspase-independent cell death. 8 More recently, GzmM-deficient mice were generated and shown to display increased susceptibility to murine cytomegalovirus (CMV) infections but displayed a normal NK and T-cell development with normal NK-mediated cytotoxicity. 9 The 5 human granzyme genes (GZMA, GZMB, GZMH, GZMK, GZMM) are clustered on 3 different chromosomes. 10 GZMA and GZMK are located on chromosome 5 and GZMM on chromosome 19. 11 GZMB and GZMH share high structural homology (71% amino acid identity) and belong to a tightly linked gene cluster on chromosome 14, which also harbors cathepsin G and mast cell chymase. Despite their high sequence homology, both enzymes bear very distinct enzymatic activities. GzmB cleaves caspase 8-like specific sequences after acidic residues. 12 In contrast, GzmH is shown to have chymotrypsin-like thioester activity with a preference for hydrophobic, aromatic amino acid residues (Phe or Tyr) at the P1 site. 2 In a recent study, Sedelies and coworkers analyzed the expression of GzmH in human blood leukocytes. 13 Using a new GzmH-specific antibody they showed the discordant regulation of GzmH and GzmB. GzmH was constitutively expressed in NK56 ϩ CD3 -NK cells irrespective of its activation status, and in contrast to GzmB was not present in activated CD8 ϩ T cells. This finding thereby suggests a pivotal role for GzmH in NK cell-induced cell death.In the rodent genomes, a highly variable number of paralogous genes were identified in the region bordered by cathepsin G and GZMB. ...

Ubiquitous calpains (-and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x L at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

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Ubiquitously expressed calpains are Ca 2؉ -dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca 2؉ -induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca 2؉ -dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo.

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