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Granule-mediated killing: pathways for granzyme B-initiated apoptosis.

Talanian RV, Yang X, Turbov J, Seth P, Ghayur T, Casiano CA, Orth K, Froelich CJ - J. Exp. Med. (1997)

Bottom Line: In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present.However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates.The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.

View Article: PubMed Central - PubMed

Affiliation: BASF Bioresearch Corporation, Worcester, Massachusetts 01605, USA.

ABSTRACT
We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.

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Blockade of caspase proteolytic activities with oligopeptide  inhibitors during GrB-induced apoptosis: effect on Ac-DEVD-afc fluorogenic activity and cleavage of death substrates, PARP, snRNP, and lamin  B. (a) z-DEVD-fmk (100 μM) inhibits Ac-DEVD-afc cleavage during  GrB/AD-mediated apoptosis. Target cells were pretreated with z-DEVD-fmk for 15 min. After exposure to GrB/AD for the times indicated, cells  were withdrawn for measurement of fluorogenic Ac-DEVD-afc activity.  (b) Generation of the apoptotic fragments of PARP, snRNP, and lamin B  is completely inhibited by the combination of z-DEVD-fmk and z-VAD-fmk. Total Jurkat cell lysates obtained from control cells and cells treated  for 1, 4, and 18 h with either GrB alone, GrB/AD, or GrB/AD plus  z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in 12%  SDS–polyacrylamide gels and proteins were transferred to nitrocellulose.  Individual lanes (containing protein corresponding to ∼106 cells) were  reacted with specific human autoantibodies to PARP, snRNP, and lamin  B. Representative blots are shown. Intact proteins are indicated by lines,  whereas proteolytic fragments are indicated by arrows. Numbers to the  right represent relative molecular weights.
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Figure 2: Blockade of caspase proteolytic activities with oligopeptide inhibitors during GrB-induced apoptosis: effect on Ac-DEVD-afc fluorogenic activity and cleavage of death substrates, PARP, snRNP, and lamin B. (a) z-DEVD-fmk (100 μM) inhibits Ac-DEVD-afc cleavage during GrB/AD-mediated apoptosis. Target cells were pretreated with z-DEVD-fmk for 15 min. After exposure to GrB/AD for the times indicated, cells were withdrawn for measurement of fluorogenic Ac-DEVD-afc activity. (b) Generation of the apoptotic fragments of PARP, snRNP, and lamin B is completely inhibited by the combination of z-DEVD-fmk and z-VAD-fmk. Total Jurkat cell lysates obtained from control cells and cells treated for 1, 4, and 18 h with either GrB alone, GrB/AD, or GrB/AD plus z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in 12% SDS–polyacrylamide gels and proteins were transferred to nitrocellulose. Individual lanes (containing protein corresponding to ∼106 cells) were reacted with specific human autoantibodies to PARP, snRNP, and lamin B. Representative blots are shown. Intact proteins are indicated by lines, whereas proteolytic fragments are indicated by arrows. Numbers to the right represent relative molecular weights.

Mentions: To allow the assessment of caspase activation by GrB in cells independently of caspase auto- and trans-activation, we explored the use of irreversible peptidic ligands for full caspase inhibition. Caspase activation in GrB/AD-treated cells was measured by cleavage of the peptidic substrate Ac-DEVD-afc, a readout of caspase-3–like proteolytic activity that is typical of lysates of cells undergoing apoptosis. On addition of GrB/AD, we observed a transient induction of caspase-3–like activity that was fully inhibited by z-DEVD-fmk at 100 μM (Fig. 2 a). To test more stringently for full inhibition of all caspases, we evaluated cell lysates for GrB/AD-induced cleavage of PARP, snRNP, and lamin B to the signature apoptotic fragments of 85, 40, and 45 kD, respectively. Because z-DEVD-fmk at 100 μM failed to block PARP cleavage completely (data not shown), we used a combination of z-DEVD-fmk and z-VAD-fmk each at 100 μM. The two inhibitors completely prevented cleavage of all three proteins up to 18 h after GrB/AD application (Fig. 2 b). We conclude that these inhibitors at 100 μM each give full caspase inhibition.


Granule-mediated killing: pathways for granzyme B-initiated apoptosis.

Talanian RV, Yang X, Turbov J, Seth P, Ghayur T, Casiano CA, Orth K, Froelich CJ - J. Exp. Med. (1997)

Blockade of caspase proteolytic activities with oligopeptide  inhibitors during GrB-induced apoptosis: effect on Ac-DEVD-afc fluorogenic activity and cleavage of death substrates, PARP, snRNP, and lamin  B. (a) z-DEVD-fmk (100 μM) inhibits Ac-DEVD-afc cleavage during  GrB/AD-mediated apoptosis. Target cells were pretreated with z-DEVD-fmk for 15 min. After exposure to GrB/AD for the times indicated, cells  were withdrawn for measurement of fluorogenic Ac-DEVD-afc activity.  (b) Generation of the apoptotic fragments of PARP, snRNP, and lamin B  is completely inhibited by the combination of z-DEVD-fmk and z-VAD-fmk. Total Jurkat cell lysates obtained from control cells and cells treated  for 1, 4, and 18 h with either GrB alone, GrB/AD, or GrB/AD plus  z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in 12%  SDS–polyacrylamide gels and proteins were transferred to nitrocellulose.  Individual lanes (containing protein corresponding to ∼106 cells) were  reacted with specific human autoantibodies to PARP, snRNP, and lamin  B. Representative blots are shown. Intact proteins are indicated by lines,  whereas proteolytic fragments are indicated by arrows. Numbers to the  right represent relative molecular weights.
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Related In: Results  -  Collection

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Figure 2: Blockade of caspase proteolytic activities with oligopeptide inhibitors during GrB-induced apoptosis: effect on Ac-DEVD-afc fluorogenic activity and cleavage of death substrates, PARP, snRNP, and lamin B. (a) z-DEVD-fmk (100 μM) inhibits Ac-DEVD-afc cleavage during GrB/AD-mediated apoptosis. Target cells were pretreated with z-DEVD-fmk for 15 min. After exposure to GrB/AD for the times indicated, cells were withdrawn for measurement of fluorogenic Ac-DEVD-afc activity. (b) Generation of the apoptotic fragments of PARP, snRNP, and lamin B is completely inhibited by the combination of z-DEVD-fmk and z-VAD-fmk. Total Jurkat cell lysates obtained from control cells and cells treated for 1, 4, and 18 h with either GrB alone, GrB/AD, or GrB/AD plus z-DEVD-fmk and z-VAD-fmk. The lysates were electrophoresed in 12% SDS–polyacrylamide gels and proteins were transferred to nitrocellulose. Individual lanes (containing protein corresponding to ∼106 cells) were reacted with specific human autoantibodies to PARP, snRNP, and lamin B. Representative blots are shown. Intact proteins are indicated by lines, whereas proteolytic fragments are indicated by arrows. Numbers to the right represent relative molecular weights.
Mentions: To allow the assessment of caspase activation by GrB in cells independently of caspase auto- and trans-activation, we explored the use of irreversible peptidic ligands for full caspase inhibition. Caspase activation in GrB/AD-treated cells was measured by cleavage of the peptidic substrate Ac-DEVD-afc, a readout of caspase-3–like proteolytic activity that is typical of lysates of cells undergoing apoptosis. On addition of GrB/AD, we observed a transient induction of caspase-3–like activity that was fully inhibited by z-DEVD-fmk at 100 μM (Fig. 2 a). To test more stringently for full inhibition of all caspases, we evaluated cell lysates for GrB/AD-induced cleavage of PARP, snRNP, and lamin B to the signature apoptotic fragments of 85, 40, and 45 kD, respectively. Because z-DEVD-fmk at 100 μM failed to block PARP cleavage completely (data not shown), we used a combination of z-DEVD-fmk and z-VAD-fmk each at 100 μM. The two inhibitors completely prevented cleavage of all three proteins up to 18 h after GrB/AD application (Fig. 2 b). We conclude that these inhibitors at 100 μM each give full caspase inhibition.

Bottom Line: In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present.However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates.The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.

View Article: PubMed Central - PubMed

Affiliation: BASF Bioresearch Corporation, Worcester, Massachusetts 01605, USA.

ABSTRACT
We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.

Show MeSH
Related in: MedlinePlus