Abstract
Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.
Highlights
Granzyme B (Gzm B)1 is a serine protease found within the lytic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells [1,2,3]
The route chosen by Gzm B to initiate caspase activation most likely depends on the effective concentration of this granzyme that is delivered by the cytotoxic T lymphocyte/natural killer (CTL/NK) into the target cell
Gzm B and cytochrome c (Cyt c)/dATP activated the same repertoire of caspases, the kinetics and hierarchy of proteolytic events clearly differed between these distinct caspase activation mechanisms
Summary
Gzm B, granzyme B; CTL, cytotoxic T lymphocyte; NK, natural killer; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; AFC, amino-4-trifluoromethylcoumarin; IPG, immobilized pH gradient; CEB, cell extract buffer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Cyt c, cytochrome c. Studies using purified Gzm B suggest that nanomolar (50 –100 nM) amounts of this granzyme are sufficient to engage the target cell death machinery [30] At these concentrations, Bid has been proposed as the preferred Gzm B substrate this has been the subject of debate [23, 31, 32]. Several caspases (caspase-3, -6, and -7) are thought to contribute to the destruction of the cell from within, probably through targeting hundreds of proteins for restricted proteolysis [33, 34] This aspect of apoptosis remains relatively poorly understood due to the sheer number of substrates that may be targeted by caspases. Using proteomic analysis we show that caspase-3 is the major target protease of granzyme B and is responsible for proteolysis of numerous cellular substrate proteins during the demolition phase of apoptosis
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