Patricia Jans

Efficient Nuclear Targeting of Granzyme B and the Nuclear Consequences of Apoptosis Induced by Granzyme B and Perforin Are Caspase-dependent, but Cell Death Is Caspase-independent

The secretory lysosomes of cytolytic lymphocytes house the principal apoptotic molecules for eliminating virus-infected cells: a membranolytic agent, perforin, and the serine protease, granzyme B. Perforin allows granzyme B access to cytosolic and nuclear substrates that, when cleaved, result in the characteristic apoptotic phenotype. Key among these substrates is a family of cytoplasmic caspases that mediate cell suicide. We have examined the caspase dependence of several nuclear and cytoplasmic parameters of apoptosis induced by purified perforin and granzyme B. Cell membrane leakage in response to perforin and granzyme B was independent of caspase activation; however, nuclear events such as DNA fragmentation and nuclear condensation and disintegration were abolished by the broad-acting caspase inhibitor, z-VAD-fmk. Despite being spared from nuclear damage, z-VAD-fmk-treated cells exposed to both cytotoxins uniformly died when they were re-cultured, while cells exposed to perforin or granzyme alone survived and proliferated as readily as untreated cells. Pretreatment of cells with z-VAD-fmk also resulted in reduced granzyme B nuclear uptake following addition of perforin; however, its uptake into the cytoplasm in the absence of perforin was unaffected. We conclude that cell death in response to perforin and granzyme B does not require caspase activation and still proceeds efficiently through non-nuclear pathways when nuclear substrate cleavage is inhibited.

Nuclear Transport of Granzyme B (Fragmentin-2) DEPENDENCE ON PERFORIN IN VIVO AND CYTOSOLIC FACTORS IN VITRO

Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei. In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semi-permeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h. In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5- and 3-fold those in the cytoplasm. In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPγS (guanosine 5′-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol. Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting. The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process. Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction.

Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.

The cytokine interleukin-5 (IL-5) effects cotransport of its receptor subunits to the nucleus in vitro

Interleukin (IL)‐5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have recently shown that human (h) IL‐5 both possesses a functional nuclear localization signal capable of targeting a heterologous protein to the nucleus and localises to the nucleus of intact receptor‐expressing cells. In this study, the extracellular domains of the hIL‐5 α‐ and β‐receptor subunits were expressed in baculovirus, fluorescently labelled and assayed for nuclear targeting in vitro in the absence and presence of IL‐5. The β‐subunit, which lacks IL‐5 binding activity, only accumulated in the nucleus in the presence of both the hIL‐5 binding α‐subunit and hIL‐5. The IL‐5‐binding α‐subunit showed similar results. IL‐5 thus effected nuclear transport of its α‐ and β‐receptor subunits apparently through a ‘piggy back’ mechanism, raising the possibility that IL‐5s nuclear signalling role may be to cotarget its receptor subunits to the nucleus. This is the first demonstration of nuclear protein piggy back transport in vitro.

A FUNCTIONAL BIPARTITE NUCLEAR LOCALISATION SIGNAL IN THE CYTOKINE INTERLEUKIN-5

Interleukin (IL)‐5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have identified a bipartite nuclear localisation signal (NLS) within amino acids 95–111 of human IL‐5 (hIL‐5), also present in mouse IL‐5 (mIL‐5). hIL‐5 and mIL‐5 were labelled fluorescently, and nuclear uptake subsequent to membrane binding and internalisation by intact receptor expressing cells visualised and quantified using confocal laser scanning microscopy. hIL‐5 and mIL‐5 were shown to be transported to the nucleus in in vivo and in vitro nuclear protein import assays. The hIL‐5 NLS was able to target a heterologous protein to the nucleus both in vivo and in vitro. Mutations within the proximal arm of the NLS abrogated nuclear targeting activity, confirming its bipartite nature. The results imply a nuclear signalling role for IL‐5 additional to pathways linked to the membrane receptor system.

Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T‐antigen NLS by importin: effect of conformation

SV40 large tumor‐antigen (T‐ag) nuclear import is enhanced by the protein kinase CK2 (CK2) site (Ser111Ser112) flanking the nuclear localization sequence (NLS). Here we use site‐directed mutagenesis to examine the influence of negative charge and conformation at the site on T‐ag nuclear import and recognition by the NLS‐binding importin subunits. Negative charge through aspartic acid in place of Ser111 simulated CK2 phosphorylation in enhancing nuclear accumulation to levels well above those of proteins lacking a functional CK2 site. This was shown to be through enhancement of T‐ag NLS recognition by importin using an ELISA‐based assay. Asp112‐substituted mutants containing proline at positions 109, 110 (wild‐type position) or 111 were compared to assess the role of conformation at the CK2 site. Maximal nuclear import of the protein with Pro109 was lower than that of the Pro110 derivative, with the Pro111 variant even lower, these differences also being attributable to effects on importin binding. All results indicate a correlation of the initial nuclear import rate with the importin binding affinity, demonstrating that NLS recognition by importin is a key rate‐determining step in nuclear import.