Perforingranzymebased cytotoxicity

1.2.1 Perforin and granzymes

The insertion of perforin into the plasma membrane of target cells facilitates the entry of granzymes inside the cell cytoplasm (5, 33). Granzymes, in turn, activate programmed cell death (34). At least 10 different serine proteases are expressed in CTL and/or NK cells, granzyme A and granzyme B being the major components of lytic granules and the main contributors to cytotoxicity (6, 34, 35). Although granzyme B is a serine protease, it shares with caspases an unusual substrate specificity characterized by the requirement of an Asp residue at the PI position. Granzyme A, on the contrary, has a tryptase-like specificity (34), suggesting functional versatility of the system. This versatility has been evidenced in granzyme A-knock-out mice, where cell-mediated cytotoxicity is normal (36), and in granzyme B-knock-out mice, where although cell-mediated cytotoxicity is blocked at short incubation times (4 h), it is completely recovered at longer times (16 h) (37).

1.2.2 Mechanism for perforin/granzymes-induced apoptosis

A possible connection between granzyme-induced cell death and caspase activation has been demonstrated, since it was observed that purified granzyme B cleaves and activates caspase-3 (38) or its close homologue caspase-7 (39). It was later demonstrated that in short-term assays (3^4- h), the peptide inhibitors Ac-DEVD-CHO (40) and Z-VAD-fmk (41) prevented nuclear apoptosis induced by CTL on Fas-negative targets, as measured by the [125I]iododeoxyuridine release assay and nuclear staining. These studies clearly implicated caspases in perforin/granzyme-based cytotoxicity exerted by CTL. However, caspase inhibitors did not prevent cell lysis under the same or similar experimental conditions, as measured by the 51Cr release assay (41, 42) or membrane manifestations of apoptosis, like phosphatidylserine exposure (41). In addition, the inhibition of CTL-induced nuclear apoptosis

FasL Fas

FADD

Membrane/cytoplasmic apoptosis

Effector caspases (CASP-3, 6 and 7)

Nucleus

^^^ Perforin o Granzymes n o Target cell O

Effector caspases (CASP-3, 6 and 7)

Nuclear apoptosis

Nucleus o Granzyme B — o Granzyme A

Membrane/cytoplasmic apoptosis

Figure 1. Schematic representation of the known biochemical mechanisms for apoptosis induction by CTL on target cells (see Section 1 for details).

by Ac-DEVD-CHO observed in 3-4 h assays was no longer observed in 6-16 h assays. The combination of Ac-DEVD-CIIO and an inhibitor of granzyme A, inhibited nuclear apoptosis at any time-point tested (40). Altogether, these data indicate that: (i) granzyme B induces the nuclear manifestations of apoptosis through a mechanism mediated by caspases; (ii) granzyme B induecs cytoplasmic apoptotic manifestations leading to cell lysis through a caspasc-independent mechanism; and (iii) all the manifestations of granzyme A-induced apoptosis, nuclear and cytoplasmic, seem to proceed through a caspase-independent mechanism. A summary of all these considerations is shown in Figure I.

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