Extracellular granzymes: current perspectives

Buzza, Marguerite S.; Bird, Phillip I.

doi:10.1515/bc.2006.106

Cited by 117 publications

(159 citation statements)

References 98 publications

(73 reference statements)

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“…Using timelapse microscopy, we confirmed that treatment of MC57s with sublytic Pfp alone or with mGzmA S-A and Pfp did not induce death or changes in cell morphology (Figure 2c). Treatment with (active) mGzmA alone resulted in target cell detachment, most likely due to GzmA-mediated cleavage of cell adhesion proteins; [14][15][16] however, the cells continued to proliferate and remained viable after 24 h ( Figures 2b and c, Supplementary Figure S2I). In contrast, treatment with mGzmA and Pfp produced the characteristic elongated morphology of GzmB À / À NK-induced alternate death ( Figure 2civ, Supplementary Movie S4).…”

Section: Resultsmentioning

confidence: 99%

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

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Section: Resultsmentioning

confidence: 99%

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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“…Purified mitochondria from Jurkat cells were treated with GrB (160 nM) for the indicated time periods. Subsequently, the mitochondria were left untreated (left; lanes [1][2][3][4][5][6] or treated with proteinase K (30 g/ml for 10 min followed by 1 M phenylmethylsulfonyl fluoride to stop the reaction; right, lanes [7][8][9][10][11][12]. The mitochondria were then spun to separate the pellets and their supernatants.…”

Section: F and H)mentioning

confidence: 99%

“…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)(2)(3)(4)(5)(6). Granzyme B (GrB) 3 exerts its cytotoxic effect after perforin-dependent intracellular delivery, acting in both cytosolic and nuclear compartments.…”

mentioning

confidence: 99%

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

Han

Goldstein

Hou

et al. 2010

Journal of Biological Chemistry

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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...

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“…Although SERPINB9 is an efficient inhibitor of gzmB inside the cell, gzmB is also known to be present and active extracellularly, where it is likely to be involved in the degradation of extracellular matrix molecules, [62][63][64][65][66][67] and may have pathophysiological functions in blood coagulation 63 and atheromatous disease (reviewed by Chamberlain and Granville 68 and elsewhere in this volume). Past investigations into potential extracellular inhibitors of gzmB have suggested a2-macroglobulin or SERPINA1 as possible candidates; 69 however, SERPINA1 shows no gzmB inhibitory activity in vitro.…”

Section: Regulation Of Extracellular Gzmbmentioning

confidence: 99%

“…However, gzmA does have a range of extracellular substrates (reviewed by Buzza and Bird 62 ) and it has been proposed that its activity in plasma is controlled by SERPINC1 (antithrombin III). 82 SERPINC1 is best characterised as an inhibitor of factor Xa and thrombin in the clotting cascade (reviewed by Pike et al 12 ), and as such it circulates at very high concentrations.…”

Section: Serpinc1 and Granzyme Amentioning

confidence: 99%

Control of granzymes by serpins

2009

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Although proteolysis mediated by granzymes has an important role in the immune response to infection or tumours, unrestrained granzyme activity may damage normal cells. In this review, we discuss the role of serpins within the immune system, as specific regulators of granzymes. The well-characterised human granzyme B-SERPINB9 interaction highlights the cytoprotective function that serpins have in safeguarding lymphocytes from granzymes that may leak from granules. We also discuss some of the pitfalls inherent in using rodent models of granzyme-serpin interactions and the ways in which our understanding of serpins can help resolve some of the current, contentious issues in granzyme biology. Cell Death and Differentiation (2010) 17, 586-595; doi:10.1038/cdd.2009; published online 6 November 2009As described elsewhere in this series, granzymes are key mediators of cytotoxic lymphocyte (CL) function, exhibiting both intracellular and extracellular functions. Thus, it is imperative that their activities be tightly controlled. Too little activity reduces the effectiveness of the immune system, resulting in infection and cancer. Conversely, overactivity can lead to disease caused by tissue destruction and cellular apoptosis. This review focusses on the manner in which granzyme activity is controlled at the posttranslational level by members of the serpin superfamily.The serpin superfamily is an ever-expanding group of structurally related proteins, currently comprising approximately 1000 members across all kingdoms of life. 1,2 Serpins function as intracellular or extracellular regulators of a wide range of physiological processes such as complement activation, blood coagulation and apoptosis. Within the vertebrate immune system, they control proteases of the classical and innate complement systems, and are also potent inhibitors of many leukocyte granule proteases. Serpin StructureStructures of almost 100 serpins from viruses to humans have been determined and all conform to the same basic architecture first described in 1984. The fold comprises nine a-helices, three b-sheets and a variable reactive centre loop (RCL), which is the primary site of interaction with target proteases (Figure 1a). The first structure determined was human SERPINA1 cleaved within the RCL. 3 Surprisingly, it was found that the residues around the cleavage point were separated by almost 70 Å , with the N-terminal portion of the RCL inserted into b-sheet A to form a fully antiparallel sixstranded sheet. It has subsequently been shown that, in the native state, the RCL is solvent exposed, and that its insertion into b-sheet A is a consequence of the inhibitory mechanism. The RCL is flanked by two highly conserved motifs (the proximal and distal hinges) that permit its insertion into b-sheet A. Insertion is also facilitated by the breach and shutter regions that assist the opening of b-sheet A and by the movement of helix F. 4 The RCL is a critical determinant of serpin inhibitory specificity, acting as a pseudosubstrate and cleavage site for the...

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Extracellular granzymes: current perspectives

Cited by 117 publications

References 98 publications

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

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

Control of granzymes by serpins

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