Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

Abstract

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrBtreated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.The granzyme family of serine proteases are pivotal mediators of apoptosis utilized by cytotoxic lymphocytes against targets such as virus-infected cells and tumor cells (1-6). Granzyme B (GrB) 3 exerts its cytotoxic effect after perforin-dependent intracellular delivery, acting in both cytosolic and nuclear compartments. The capacity of GrB to initiate a mitochondrial apoptotic cascade that features permeabilization of the mitochondrial outer membrane and release of apoptogenic proteins has been tied directly to cleavage of Bid (7-10). GrB-generated tBid activates the downstream executioners of the mitochondrial apoptotic cascade, Bax and/or Bak (11). However, mitochondria of Bid-deficient cells continue to release cytochrome c in response to GrB, suggesting the existence of an alternative mechanism(s). Our recent studies demonstrated that GrB induces a mitochondrial apoptotic cascade by cleaving Mcl-1, an antiapoptotic Bcl-2 family member that resides on the mitochondrial outer membrane in a complex with a potent proapoptotic BH3-only protein, Bim. GrB-mediated degradation of Mcl-1 releases sequestered Bim, allowing the execution of a distinct apoptotic cascade (12, 13). The mitochondrial function of BH3-only proteins, including Bid and Bim, is strictly Bax/Bak-dependent (14). Accordingly, Bax/Bak-deficient cells are relatively resistant to GrB-mediated apoptosis, but mitochondria continue to undergo significant depolarization that cannot be attributed to currently described mechanisms of GrB-mediated apoptosis (10,15,16).Mitochondrial polarization (i.e. maintenance of its inner membrane potential) is ulti...

Granzyme B-induced mitochondrial ROS are required for apoptosis

“…Tagged complex I subunits overexpressed in the B-cell line 721.221, or MEF localized to the mitochondria and integrated into complex I (Supplementary Figures 1e-g and data not shown). GB induced a dose-and time-dependent cleavage of tagged NDUFV1, NDUFS2 and NDUFS1 similar to PARP-1 and HAX-1 11,12 cleavage (Supplementary Figures 2a-c). GB did not cleave NDUFS3, NDUFS7 or NDUFA13 (data not shown).…”

“…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][12][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.…”

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

“…[5][6][7][8][9][10] GB-induced cell death also involves Bid/Bax/ Bak-mediated mitochondrial outer membrane permeabilization for the release of the apoptogenic factors cytochrome c, Smac/Diablo, Endo G, HtrA2 and AIF. 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.…”

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“…HAX1 is also subject to cleavage by granzyme B. 25 Whereas the N-terminal cleavage product localizes to mitochondria and mediates depolarization, the C-terminal cleavage product can be found in the cytosol. 25 HAX1 may interact with X-linked inhibitor of apoptosis, preventing polyubiquitination of X-linked inhibitor of apoptosis 1 and thus act to maintain viability.…”

“…25 Whereas the N-terminal cleavage product localizes to mitochondria and mediates depolarization, the C-terminal cleavage product can be found in the cytosol. 25 HAX1 may interact with X-linked inhibitor of apoptosis, preventing polyubiquitination of X-linked inhibitor of apoptosis 1 and thus act to maintain viability. 26 In addition to a prominent role in protecting against cell death, HAX1 has also been reported to be involved in multiple other cellular functions, such as calcium homeostasis, cell motility and cytoskeletal rearrangement, and messenger RNA processing.…”