Abstract
Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as promotes cytochrome c (cyt c) release and loss of mitochondrial membrane potential. We investigated how GrB affects mitochondria utilizing an in vitro cell-free system and determined that cyt c release and permeability transition are initiated by distinct mechanisms. The cleavage of cytosolic BID by GrB results in truncated BID, initiating mitochondrial cyt c release. BID is the sole cytosolic protein responsible for this phenomenon in vitro, yet caspases were found to participate in cyt c release in some cells. On the other hand, GrB acts directly on mitochondria in the absence of cytosolic S100 proteins to open the permeability transition pore and to disrupt the proton electrochemical gradient. We suggest that GrB acts by two distinct mechanisms on mitochondria that ultimately lead to mitochondrial dysfunction and cellular demise.
Highlights
Granzyme B (GrB) is an aspartyl serine protease located in the granules of cytotoxic T cells (CTL) and natural killer (NK) cells that induce apoptosis
We will demonstrate that GrB mediates cytochrome c (CC) release from isolated mitochondria in vitro through an S100 protein that we identify as the BCL-2 family member BID
We determined in a cell free assay that GrB-mediated CC release requires a protein in the cytosolic S100 fraction (Fig.1A)
Summary
Granzyme B (GrB) is an aspartyl serine protease located in the granules of cytotoxic T cells (CTL) and natural killer (NK) cells that induce apoptosis. Recent work suggests that mitochondria play a key role in the regulation of cell death in mammalian cells through the release of cytochrome c (CC), as well as the loss of the mitochondrial membrane potential (∆Ψm). GrB has been shown to induce mitochondria to release CC that interacts with Apaf-1 and caspase 9 resulting in the activation of effector caspases and the subsequent apoptotic biochemical and morphological cellular changes (2,3). We will demonstrate that GrB mediates CC release from isolated mitochondria in vitro through an S100 protein that we identify as the BCL-2 family member BID. GrB in the absence of BID or other S100 proteins can open the mitochondrial PT pore with the loss of ∆Ψm. We have identified two mechanisms by which GrB disrupts mitochondrial function and promotes cell death
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