scholarly journals Activation of Membrane-associated Procaspase-3 Is Regulated by Bcl-2

1999 ◽  
Vol 144 (5) ◽  
pp. 915-926 ◽  
Author(s):  
Joseph F. Krebs ◽  
Robert C. Armstrong ◽  
Anu Srinivasan ◽  
Teresa Aja ◽  
Angela M. Wong ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Caspase 3 ◽  
Caspase Activity ◽  
Intracellular Membrane ◽  
Caspase Activation ◽  
Intracellular Membranes ◽  
Membrane Bound ◽  
Spontaneous Activation ◽  
Active Caspase

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2–inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

scholarly journals A Functional NADPH Oxidase Prevents Caspase Involvement in the Clearance of Phagocytic Neutrophils

2007 ◽  
Vol 75 (7) ◽  
pp. 3256-3263 ◽  
Author(s):  
Rachel P. Wilkie ◽  
Margret C. M. Vissers ◽  
Mike Dragunow ◽  
Mark B. Hampton
Keyword(s):  
Nadph Oxidase ◽  
Caspase 3 ◽  
Caspase Activity ◽  
Wild Type ◽  
Caspase Activation ◽  
Effector Caspases ◽  
Nadph Oxidase Complex ◽  
Normal Human ◽  
Active Caspase

ABSTRACT Neutrophils play a prominent role in host defense. Phagocytosis of bacteria leads to the formation of an active NADPH oxidase complex that generates reactive oxygen species for bactericidal purposes. A critical step in the resolution of inflammation is the uptake of neutrophils by macrophages; however, there are conflicting reports on the mechanisms leading to the apoptosis of phagocytic neutrophils. The aim of this study was to clarify the role of effector caspases in these processes. Caspase activity was measured by DEVDase activity assays or immunofluorescence detection of active caspase-3. With normal human and wild-type murine neutrophils there was no caspase activation following phagocytosis of Staphylococcus aureus. However, caspase activity was observed in phagocytic neutrophils with a defective NADPH oxidase, including neutrophils isolated from X-linked gp91phox knockout chronic granulomatous disease mice. These results indicate that a functional NADPH oxidase and the generation of oxidants in the neutrophil phagosome prevent the activation of the cytoplasmic caspase cascade.

scholarly journals Sorcin modulates mitochondrial Ca2+ handling and reduces apoptosis in neonatal rat cardiac myocytes

2013 ◽  
Vol 304 (3) ◽  
pp. C248-C256 ◽  
Author(s):  
Jorge Suarez ◽  
Patrick M. McDonough ◽  
Brian T. Scott ◽  
Angelica Suarez-Ramirez ◽  
Hong Wang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cardiac Myocytes ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Permeabilized Cells ◽  
Neonatal Rat Cardiac Myocytes ◽  
Rat Cardiac Myocytes ◽  
Uptake And Release

Sorcin localizes in cellular membranes and has been demonstrated to modulate cytosolic Ca2+ handling in cardiac myocytes. Sorcin also localizes in mitochondria; however, the effect of sorcin on mitochondrial Ca2+ handling is unknown. Using mitochondrial pericam, we measured mitochondrial Ca2+ concentration and fluxes in intact neonatal cardiac myocytes overexpressing sorcin. Our results showed that sorcin increases basal and caffeine-stimulated mitochondrial Ca2+ concentration. This effect was associated with faster Ca2+ uptake and release. The effect of sorcin was specific for mitochondria, since similar results were obtained with digitonin-permeabilized cells, where cytosolic Ca2+ flux was disrupted. Furthermore, mitochondria of cardiac myocytes in which sorcin was overexpressed were more Ca2+-tolerant. Experiments analyzing apoptotic signaling demonstrated that sorcin prevented 2-deoxyglucose-induced cytochrome c release. Furthermore, sorcin prevented hyperglycemia-induced cytochrome c release and caspase activation. In contrast, antisense sorcin induced caspase-3 activation. Thus, sorcin antiapoptotic properties may be due to modulation of mitochondrial Ca2+ handling in cardiac myocytes.

scholarly journals Caspase activation contributes to endotoxin-induced diaphragm weakness

2006 ◽  
Vol 100 (6) ◽  
pp. 1770-1777 ◽  
Author(s):  
Gerald S. Supinski ◽  
Leigh A. Callahan
Keyword(s):  
Caspase 3 ◽  
Muscle Force ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Respiratory Muscle Weakness ◽  
Protein Levels ◽  
Diaphragmatic Weakness ◽  
Caspase Protein ◽  
Diaphragm Weakness ◽  
Active Caspase

Infections produce significant respiratory muscle weakness, but the mechanisms by which inflammation reduces muscle force remain incompletely understood. Recent work suggests that caspase 3 releases actin and myosin from the contractile protein lattice, so we postulated that infections may reduce skeletal muscle force by activating caspase 3. The present experiments were designed to test this hypothesis by determining 1) diaphragm caspase 3 activation in the diaphragm after endotoxin and 2) the effect of a broad-spectrum caspase inhibitor, Z-Val-Ala-Asp(OCH3)-fluoromethylketone (zVAD-fmk), and a selective caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO), on endotoxin-induced diaphragm weakness. Caspase 3 activation was assessed by measuring caspase protein levels and by measuring cleavage of a fluorogenic substrate. Diaphragm force was measured in response to electrical stimulation (1–150 Hz). Caspase-mediated spectrin degradation was assessed by Western blotting. Parameters were compared in mice given saline, endotoxin (12 mg/kg ip), endotoxin plus zVAD-fmk (3 mg/kg iv), zVAD-fmk alone, or endotoxin plus DEVD-CHO (3 mg/kg iv). Endotoxin increased diaphragm active caspase 3 protein ( P < 0.003), increased caspase 3 activity ( P < 0.002), increased diaphragm spectrin degradation ( P < 0.001), and reduced diaphragm force ( P < 0.001). Administration of zVAD-fmk or DEVD-CHO prevented endotoxin-induced weakness (e.g., force in response to 150-Hz stimulation was 23.8 ± 1.4, 12.1 ± 1.3, 23.5 ± 0.8, 22.7 ± 1.3, and 24.4 ± 0.8 N/cm2, respectively, for control, endotoxin, endotoxin plus zVAD-fmk, endotoxin plus DEVD-CHO, and zVAD-fmk alone treated groups, P < 0.001). Caspase inhibitors also prevented spectrin degradation. In conclusion, endotoxin administration elicits significant diaphragm caspase 3 activation and caspase-mediated diaphragmatic weakness.

scholarly journals Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia–reoxygenation

2000 ◽  
Vol 347 (3) ◽  
pp. 669-677 ◽  
Author(s):  
Zheng DONG ◽  
Pothana SAIKUMAR ◽  
Yogendra PATEL ◽  
Joel M. WEINBERG ◽  
Manjeri A. VENKATACHALAM
Keyword(s):  
Cytochrome C ◽  
Serine Protease ◽  
Protease Inhibitors ◽  
Caspase 3 ◽  
Rat Kidney ◽  
Serine Protease Inhibitors ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Chloromethyl Ketone ◽  
Hypoxia Reoxygenation

We have shown that reoxygenation of hypoxic rat kidney proximal tubule cells leads to apoptosis. This is mediated by translocation of Bax from the cytosol to mitochondria, accompanied by release of mitochondrial cytochrome c (cyt.c). The present study has examined the proteolytic mechanisms responsible for apoptosis during hypoxia-reoxygenation. Caspases were activated during hypoxia, as shown by cleavage of fluorogenic peptide substrates. By 5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. This was accompanied by specific processing of pro-caspase-3, -8 and -9 into active forms. Caspase activation during hypoxia was blocked by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed by the chymotryptic inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF), and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activation by these compounds resulted in arrest of apoptosis. On the other hand, the serine protease inhibitors did not prevent release of mitochondrial cyt.c during hypoxia, suggesting that these compounds blocked a critical step in post-mitochondrial caspase activation. Further studies using an in vitro reconstitution model showed that cyt.c/dATP stimulated caspase-9 processing and downstream caspase activation were significantly suppressed in the presence of TPCK and APF. Based on these results, we speculate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator, caspase-9.

scholarly journals Mild Hypothermia Reduces Apoptosis of Mouse Neurons In vitro Early in the Cascade

2002 ◽  
Vol 22 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Lijun Xu ◽  
Midori A. Yenari ◽  
Gary K. Steinberg ◽  
Rona G. Giffard
Keyword(s):  
Cytochrome C ◽  
Neuronal Apoptosis ◽  
Caspase 3 ◽  
Mild Hypothermia ◽  
Early Time ◽  
Caspase Activation ◽  
Hsp 70 ◽  
Kinase Activation ◽  
Recent Experimental Work

Recent experimental work has shown that hypothermia with even small decreases in temperature is broadly neuroprotective, but the mechanism of this protection remains unclear. Although reduction of metabolism could explain protection by deep hypothermia, it does not explain the robust protection found with mild hypothermia. Several reports have suggested that ischemic apoptosis is reduced by hypothermia. The authors examined the effects of hypothermia on neuronal apoptosis using serum deprivation, a well-accepted model that induces neuronal apoptosis. Mild hypothermia (33°C) significantly reduced the number of morphologically apoptotic neurons to less than half the number seen in normothermic culture temperatures (37°C) after 48 hours. They examined the effect of hypothermia on several steps in the cascade. Caspase-3, −8, and −9 activity was significantly increased after 24 hours at 37°C, and was significantly lower in cultures deprived of serum at 33°C. Cytochrome c translocation was reduced by hypothermia. Western blot analysis failed to detect significant changes in Bax, bcl-2, or hsp-70 at early time points, whereas hypothermia significantly reduced cJun N-terminal kinase activation. The authors conclude that small decreases in temperature inhibit apoptosis very early, possibly at the level of the initiation of apoptosis, as suggested by reduced cJun N-terminal kinase activation and before the translocation of cytochrome c, with subsequent prevention of caspase activation.

scholarly journals Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death

2003 ◽  
Vol 160 (4) ◽  
pp. 577-587 ◽  
Author(s):  
Murray C.H. Clarke ◽  
John Savill ◽  
David B. Jones ◽  
Brendon S. Noble ◽  
Simon B. Brown
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Progenitor Cell ◽  
Caspase 3 ◽  
Transmembrane Potential ◽  
Blood Platelets ◽  
Apoptotic Bodies ◽  
Caspase 9 ◽  
Daughter Cells ◽  
Membrane Bound

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

scholarly journals Biphasic Cytochrome c Release After Transient Global Ischemia and its Inhibition by Hypothermia

2005 ◽  
Vol 25 (9) ◽  
pp. 1119-1129 ◽  
Author(s):  
Heng Zhao ◽  
Midori A Yenari ◽  
Danye Cheng ◽  
Robert M Sapolsky ◽  
Gary K Steinberg
Keyword(s):  
Cerebral Ischemia ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Global Ischemia ◽  
Second Phase ◽  
Ischemic Damage ◽  
Caspase Activity ◽  
Cytochrome C Release ◽  
Western Blots ◽  
Transient Global Ischemia

Hypothermia is effective in preventing ischemic damage. A caspase-dependent apoptotic pathway is involved in ischemic damage, but how hypothermia inhibits this pathway after global cerebral ischemia has not been well explored. It was determined whether hypothermia protects the brain by altering cytochrome c release and caspase activity. Cerebral ischemia was produced by two-vessel occlusion plus hypotension for 10 mins. Body temperature in hypothermic animals was reduced to 33°C before ischemia onset and maintained for 3 h after reperfusion. Western blots of subcellular fractions revealed biphasic cytosolic cytochrome c release, with an initial peak at about 5 h after ischemia, which decreased at 12 to 24 h, and a second, larger peak at 48 h. Caspase-3 and −9 activity increased at 12 and 24 h. A caspase inhibitor, Z-DEVD-FMK, administered 5 and 24 h after ischemia onset, protected hippocampal CA1 neurons from injury and blocked the second cytochrome c peak, suggesting that caspases mediate this second phase. Hypothermia (33°C), which prevented CA1 injury, did not inhibit cytochrome c release at 5 h, but reduced cytochrome c release at 48 h. Caspase-3 and −9 activity was markedly attenuated by hypothermia at 12 and 24 h. Thus, biphasic cytochrome c release occurs after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase of cytochrome c release, possibly by blocking caspase activity.

scholarly journals Knock Out of Cell Death Pathway Components Results in Differential Caspase Expression in Response to HCV Infection

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 144
Author(s):  
Hannah L. Wallace ◽  
Lingyan Wang ◽  
Cassandra Davidson ◽  
Vipin Chelakkot ◽  
Michael Grant ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cross Talk ◽  
Caspase 3 ◽  
Caspase Activation ◽  
Knock Out ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Infected Cells ◽  
Active Caspase

Introduction: Pyroptosis (inflammatory programmed cell death) is induced after the activation of an inflammasome, ultimately resulting in pore formation and cell lysis. One factor in the pathology associated with chronic hepatitis C virus (HCV) infection is non-inflammatory caspase-3-mediated apoptosis. Our lab has found both apoptosis and pyroptosis occurring in HCV-infected Huh-7.5 cells. In the context of some viral infections, pyroptosis is beneficial to the virus; for others, pyroptosis is believed to represent an innate antiviral response. This study aimed to test the effects of knocking out components of the inflammasome pathway on caspase activation in HCV-infected cells. Methods: FAM-FLICA (Carboxyfluorescein - Fluorochrome Inhibitor of Caspases) probes or antibodies were used to visualize active caspase-1 and active caspase-3 in vitro. Huh-7.5 cells with components of the pyroptotic or apoptotic pathways knocked out (NLRP3, GSDM-D or caspase-3) were used to determine the effects of their absence on the virus and caspase activation using confocal microscopy and flow cytometry. Results: Increased levels of caspase-1 were consistently observed in HCV-infected cells compared to those in uninfected cells, and these levels increased with subsequent days post-infection. The inhibition of inflammasome activation using knock out cell lines induced the differential activation of caspase-1 and caspase-3, with the inhibition of pyroptosis, resulting in a trend towards greater expression of caspase-3, indicative of apoptosis. The inhibition of NLRP3 did not fully stop caspase-1 activation, but it was decreased. The flow cytometry results revealed a small sub-set of cells positive for both caspase-1 and caspase-3. Conclusions: These data confirm the occurrence of pyroptosis in HCV-infected cells and demonstrate the involvement of the NLRP3 inflammasome, although other inflammasome sensors might be involved. Since the inhibition of one cell death pathway resulted in the increased activation of the other, along with the presence of double-positive cells, there may be cross-talk between apoptotic and pyroptotic pathways; the role of this cross-talk during infection remains to be elucidated.

Cytochrome C Related Caspase Activation Determines Treatment Response in Childhood B- Precursor ALL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 990-990
Author(s):  
Lueder H. Meyer ◽  
Leonid Karawajew ◽  
Martin Schrappe ◽  
Wolf D. Ludwig ◽  
Klaus M. Debatin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cytochrome C ◽  
Treatment Response ◽  
Prognostic Value ◽  
Caspase 3 ◽  
Initial Response ◽  
Leukemia Cells ◽  
Induction Treatment ◽  
Caspase Activation ◽  
Cytochrome C Release

Abstract Drug resistance and treatment failure in acute leukemia may be attributed to apoptosis resistance in leukemia cells since defects in apoptosis signal transduction are commonly acquired during malignant transformation. Expression analysis of single molecules with regard to clinical outcome could so far not identify common apoptosis defects with prognostic value in primary acute leukemias. We addressed the role of apoptosis signaling for the initial response to induction treatment in pediatric B precursor lymphoblastic leukemia by functional assays. Apoptosis signaling in response to survival factor withdrawal was assessed in 82 primary leukemia samples by measurement of the key apoptotic events, caspase-3 activation and mitochondrial cytochrome c release. Caspase-3 activation directly correlated to the initial response to treatment assessed by the percentage of leukemia cells in bone marrow on day 15 (p = 0.015). Intact apoptosis signaling was indicated by a significant correlation of caspase activation and cytochrome c release was found especially in the groups of good responding patients (rs 0.375 – 0.502; p = 0.001). Interestingly, this correlation was completely absent in all groups of poor responding patients defined by insufficient leukemia cell reduction on day 8, 15 or 33. The efficacy of apoptosis signal induction in the individual leukemia is expressed by the parameter CRAC (Cytochrome c Related Activation of Caspase-3), relating the extent of caspase activation to cytochrome c release in a single patient sample. Ten of twelve patients with prednisone poor response (chi square, p= 0.031) and all patients not achieving remission on day 33 (4 of 4) had negative CRAC values, indicating deficient cytochrome c related caspase activation. Furthermore, the CRAC negative group revealed significant higher percentages of leukemia cells in bone marrow on day 15 (n=37, mean 22.8) than the CRAC positive patients (n=30, mean 6.2, p = 0.004). In addition all four relapse patients showed negative CRAC values indicating a prognostic value beyond initial treatment response. The data show that the intact relation of cytochrome c release and caspase activation indicating an intact apoptosome function is important for efficient remission induction treatment in childhood B precursor ALL. Assessment and quantification of this relation for individual patients by the parameter CRAC may serve as potential factor for treatment stratification.

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