Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.     

Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion

Reperfusion after myocardial ischemia is associated with a rapid influx of calcium, leading to activation of various enzymes including calpain. Isolated perfused adult rabbit hearts subjected to global ischemia and reperfusion were studied. Calpain or a calpain-like activity was activated within 15 min after reperfusion, and preconditioning suppressed calpain activation. In contrast, caspase activation was not detected although cytochrome c was released after ischemia and reperfusion. The pro-apoptotic BH3-only Bcl-2 family member, Bid, was cleaved during ischemia/reperfusion in the adult rabbit heart. Recombinant Bid was cleaved by calpain to a fragment that was able to mediate cytochrome c release. The calpain cleavage site was mapped to a region within Bid that is extremely susceptible to proteolysis. These findings suggest that there is cross-talk between apoptotic and necrotic pathways in myocardial ischemia/reperfusion injury.     

Inhibition of calpain but not caspase protects the testis against injury after experimental testicular torsion of rat

Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of alpha-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed alpha-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.     

Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.     

Reperfusion,not simulated ischemia,initiates intrinsic apoptosis injury in chick cardiomyocytes

Although ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of cytochrome c release during I/R and whether subsequent caspase activation contributes to reperfusion injury in confluent chick cardiomyocytes. One-hour simulated ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged ischemia for 4 h had only marginally increased cell death and no detectable cytochrome c release into the cytosol. Caspase activation could not be detected after ischemia only, but it significantly increased after reperfusion. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion oxidants initiate cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.     

Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both mu- and m-calpain activities. Capn4(-/-) MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4(-/-) MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor alpha-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4(-/-) MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4(-/-) MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor alpha- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.     

Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.Key words: curcumin, mitochondria, cytochrome c, Apaf-1, caspase, p21     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 +/- 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH(2)F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.     

高铁血红素对心肌缺血／再灌注损伤的防护作用及其机制

目的：在大鼠急性心肌缺血／再灌ii（I／R）模型上,观察高铁血红素在钙激活中性蛋白酶（calpain）介导的心肌I／R损伤中的作用。并初步探讨其可能的机制。方法：64只雄性SD大鼠随机8组（n：8）：假手术组（sham组）、（I／R）组、MDL28170＋I／R组、单纯MDL28170组、高铁血红素＋I／R组、单纯高铁血红素组、锌原卟啉Ⅸ＋高铁血红素＋I／R组、单纯锌原卟啉Ⅸ组。采用大鼠离体心脏Langendorff灌流技术,心脏I／R后,测定左室发展压（LVDP）、心肌梗死面积、冠脉流出液中的乳酸脱氢酶（LDH）释放量。检测calpain、血红素氧化酶（HO）、和半胱氨酸天冬氨酸蛋白酶3（caspase3）活性。Westernblot观察心肌钙蛋白酶抑制蛋白（calpastatin）蛋白表达。结果：①心肌I／R后,calpain、caspase3活性明显增高。calpain抑制剂MDL28170可抑制I／R诱导的LDH释放量增加,增高LVDP,缩小心肌梗死面积。②与单纯I／R组相比,大鼠预先给予高铁血红素后,心脏HO-1活性增加,calpain和caspase3活性下降。同时,LDH释放量减少,LVDP明显增高,心肌梗死面积缩小。③I／R组心肌calpastatin表达量明显低于对照组,高铁血红素组大鼠calpastatin表达量增高。HO-1的抑制剂锌原卟啉Ⅸ可取消高铁血红素对calpastain表达量的影响,并取消其心肌保护作用。结论：高铁血红素预处理可通过抑制calpain的激活,减轻大鼠心肌I／R损伤,其机制可能与增加calpastatin蛋白表达有关。     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55–60%) and calcium-dependent (40–45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.     

PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER-mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER-mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER-mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.