Granzyme B-induced mitochondrial ROS are required for apoptosis

Abstract

Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GBmediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis. Cell Death and Differentiation (2015) 22, 862-874; doi:10.1038/cdd.2014.180; published online 31 October 2014To induce cell death, human granzyme B (GB) activates effector caspase-3 or acts directly on key caspase substrates, such as the proapoptotic BH3 only Bcl-2 family member Bid, inhibitor of caspase-activated DNase (ICAD), poly-(ADPribose) polymerase-1 (PARP-1), lamin B, nuclear mitotic apparatus protein 1 (NUMA1), catalytic subunit of the DNAdependent protein kinase (DNA-PKcs) and tubulin. [1][2][3] Consequently, caspase inhibitors have little effect on human GB-mediated cell death and DNA fragmentation. 2 GB causes reactive oxygen species (ROS) production, dissipation of the mitochondrial transmembrane potential (ΔΨm) and MOMP, which leads to the release of apoptogenic factors such as cytochrome c (Cyt c), HtrA2/Omi, endonuclease G (Endo G), Smac/Diablo and apoptosis-inducing factor, from the mitochondrial intermembrane space to the cytosol. [4][5][6][7][8][9][10][11] Interestingly, cells deficient for Bid, Bax and Bak are still sensitive to human GB-induced cell death, 5,11-13 suggesting that human GB targets the mitochondria in another way that needs to be characterized. Altogether, much attention has been focused on the importance of MOMP in the execution of GB-mediated cell death, leaving unclear whether ROS production is a bystander effect or essential to the execution of GBinduced apoptosis. The mitochondrial NADH: ubiquinone oxidoreductase complex I is a key determinant in steadystate ROS production. This 1 MDa complex, composed of 44 subunits, 14 couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons to generate the ΔΨm. The importance of ROS has been previously demonstrated for caspase-3 and granzyme A (GA) pathways through the cleavage of NDUFS1 and NDUFS3, respectively. [15][16][17][18] GA induces cell death in a Bcl-2-insensitive and caspase-and MOMP-indepen...

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…Moreover, cells knocked down for Tob55/Sam50 still experienced Bid and PARP-1 cleavage (Figure 4e) when treated with GB and P; however, the release of cytochrome c was reduced in Tob55/Sam50-depleted cells (Figure 4f), indicating that GB mitochondrial entry is needed for apoptogenic factor release, most likely following the production of ROS. 18,51 GA, GM and caspase-3 induce cell death in a Tob55/ Sam50-dependent manner. Similar to GB, GA localizes in the mitochondrial matrix compartment (Figure 5a), enters the yeast mitochondria independently of Tom40 (Figure 5b) and its import was neither affected by saturation of the canonical mitochondrial import machinery with B2Δ19-DHFR in the presence of MTX (Supplementary Figures 8A and B) nor by the loss of Tom70, Tom22 and Tom20 ( Supplementary Figures 8C and D).…”

“…Additionally, we also reported that GB disrupts the mitochondrial cristae junction, possibly contributing to GB forceful passage across the inner membrane. 18 Additional experiments are needed to test this possibility.…”

“…18 Mitochondrial ROS resulted from GB-mediated cleavage of NDUFV1, NDUFS1 and NDUFS2, three subunits of mitochondrial complex I. 18 How GB that does not possess a mitochondrial targeting sequence enters the mitochondria is not known. The mitochondrial nucleoid encodes only for 13 structural proteins, while the rest of the mitochondrial proteome is nuclear-encoded and transported to the mitochondria through a sophisticated protein import machinery.…”

“…1,[9][10][11][12][13][14][15][16][17] We have recently reported that mitochondrial reactive oxygen species (ROS) have a critical role in GB pathway by amplifying apoptogenic factor release, oligonucleosomal DNA fragmentation and lysosomal membrane rupture. 18 Mitochondrial ROS resulted from GB-mediated cleavage of NDUFV1, NDUFS1 and NDUFS2, three subunits of mitochondrial complex I. 18 How GB that does not possess a mitochondrial targeting sequence enters the mitochondria is not known.…”

Gene Expression Profiling of H9c2 Cells Subjected to H₂O₂-Induced Apoptosis with/without AF-HF001

“…Mitochondrial complex I (reduced nicotinamide adenine dinucleotide (NADH) ubiquinone oxidoreductase) initiates the mitochondrial electron transport chain, and a decrease in the activity of mitochondrial complex I is associated with an increase in ROS production. 8) Jacquemin et al 9) modified the mitochondria by cleaving the Ndufs2 subunit to trigger ROS-dependent apoptosis. Massoz et al 10) reported that Ndufs3 was required for complex I activity, and Ndufs3 RNA interference reduced intracellular ROS production.…”