Perforin and graB synergize to cause apoptosis

In early studies, purified perforin was demonstrably a potent lysin (particularly for erythrocytes) but by itself was unable to reproduce apoptotic features such as blebbing of the cell membrane, DNA fragmentation and nuclear collapse in nucleated cells. Paradoxically, perforin-deficient mice first produced by Kagi and Hengartner exhibited normal T cell development and cytokine secretion but were immunodeficient due to a markedly reduced ability to induce target cell apoptosis. Kagi's observations were consistent with the hypothesis advanced by Greenberg and his colleagues that the combination of graB (fragmentin-2) and tiny (sublytic) doses of perforin could synergize to produce DNA fragmentation. Greenberg indeed demonstrated that cells exposed to just these two purified molecules underwent apoptosis. Like perforin, graB applied alone to cells did not cause apoptosis. The requirement for an 'Asp-ase' specificity was demonstrated in that proteolytically inactive graB could not produce apoptosis, while other granzymes with non-Asp-ase activities (fragmentins 1 and 3, both of which possess trypsin-like activity) produced far slower apoptotic changes in combination with perforin.

Over the past several years, the mechanism of granzyme/perforin synergy has been the topic of intense investigation. Target cells loaded with protease inhibitors such as aprotinin could withstand apoptosis by CTLs, suggesting that graB acted intra-cellularly. This was confirmed by our own studies with purified fluorescein-conjugated graB which showed that cells co-exposed to perforin rapidly took up graB into the cytoplasm and ultimately also into the nucleus. These results were further supported by immunoelectron microscopy, which demonstrated graB within cytoplasmic vacuoles and free in the cytosol, while perforin staining was confined to the target cell membrane. Because it can form membrane pores, perforin had long been postulated to permit the diffusion of toxins such as granzymes into target cells. However this 'simple diffusion' model seems unlikely to be valid, as cells exposed to graB alone became vacuolated and contained cytoplasmic graB. Furthermore, graB/perforin-induced apoptosis could not be reproduced by substituting alternative pore-forming and membranolytic agents (including complement) for perforin. Overall, these findings suggested a highly specific requirement for graB

which cleaves intracellular substrates and the generation of an equally essential but yet undefined signal at the cell membrane by perforin.

As perforin and graB can kill a very broad variety of cells, it is likely that the signal generated by perforin utilizes a pathway present in virtually all cells. Whether the uptake of graB is receptor-mediated and/or due to endocytosis remains unclear. GraA has been shown to cleave the thrombin receptor and possibly other similar, unidentified cell surface receptors. Perforin, by contrast, undergoes a change in its tertiary structure from a globular to an extended configuration in the presence of calcium ions and binds to phosphorylcholine moieties in natural (and artificial) membranes to form oligomeric polyper-forin clusters consisting of up to 18 monomers.

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